KR20130103499A - Ligands for antibody and fc-fusion protein purification by affinity chromatography - Google Patents

Abstract

식 중,
Sp는 스페이서 기(spacer group)이고;
v는 0 또는 1이고;
Am은 아미드 기 -NR¹-C(O)-이고, 여기서 NR¹이 Ar¹에 부착되고 -C(O)-가 Ar²에 부착되거나, 또는 -C(O)-가 Ar¹에 부착되고 NR¹이 Ar²에 부착되고;
R¹은 수소 또는 C₁ 내지 C₄ 알킬, 바람직하게는 수소 또는 메틸이고, 더 바람직하게는 수소이고;
Ar¹은 이가 5- 또는 6-원 치환 또는 비치환 방향족 고리이고;
Ar²는 (a) 단일 결합을 통해 추가 5- 또는 6-원 방향족 고리에 부착된 5- 또는 6-원 헤테로환 방향족 고리이거나; 또는 (b) 다환고리계의 부분으로서 추가 5- 또는 6-원 방향족 고리에 융합된 5- 또는 6-원 헤테로환 방향족 고리이거나; 또는 (c) C₁ 내지 C₄ 알킬, C₂ 내지 C₄ 알케닐, C₂ 내지 C₄ 알키닐, 할로겐, C₁ 내지 C₄ 할로알킬, 히드록실-치환된 C₁ 내지 C₄ 알킬, C₁ 내지 C₄ 알콕시, 히드록실-치환된 C₁ 내지 C₄ 알콕시, C₁ 내지 C₄ 알킬아미노, C₁ 내지 C₄ 알킬티오, 및 이의 조합으로부터 선택된 하나 이상의 치환기에 부착된 5- 또는 6-원 헤테로환 방향족 고리임.The present invention relates to the use of a ligand-substituted matrix comprising a support material and at least one ligand covalently attached to the support material, for affinity purification of an antibody or antibody fragment, wherein the ligand is represented by the following formula (I): Is indicated by:
Formula (I)

Wherein,
Sp is a spacer group;
v is 0 or 1;
Am is an amide group -NR ¹ -C (O)-, wherein NR ¹ is attached to Ar ¹ and -C (O)-is attached to Ar ² , or -C (O)-is attached to Ar ¹ NR ¹ is attached to Ar ² ;
R ¹ is hydrogen or C ₁ to C ₄ alkyl, preferably hydrogen or methyl, more preferably hydrogen;
Ar ¹ is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring;
Ar ² is (a) a 5- or 6-membered heterocyclic aromatic ring attached to a further 5- or 6-membered aromatic ring via a single bond; Or (b) a 5- or 6-membered heterocyclic aromatic ring fused to an additional 5- or 6-membered aromatic ring as part of a polycyclic ring system; Or (c) C ₁ to C ₄ alkyl, C ₂ to C ₄ alkenyl, C ₂ to C ₄ alkynyl, halogen, C ₁ To C ₄ haloalkyl, hydroxyl-substituted C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, hydroxyl-substituted C ₁ to C ₄ alkoxy, C ₁ to C ₄ alkylamino, C ₁ to C ₄ 5- or 6-membered heterocyclic aromatic ring attached to at least one substituent selected from alkylthio, and combinations thereof.

Description

LIGANDS FOR ANTIBODY AND Fc-FUSION PROTEIN PURIFICATION BY AFFINITY CHROMATOGRAPHY}

The present invention relates to the field of protein separation by affinity separation techniques, in particular chromatography using small molecule ligands, and the purification of monoclonal and polyclonal antibodies and fusion proteins, preferably containing immunoglobulin Fc moieties.

Immunoglobulins are a group of soluble proteins found in the body fluids of humans and other vertebrates. They are also referred to as "antibodies" and play an important role in the process of recognition, binding and attachment of cells. Antibodies are oligomeric glycoproteins that play an important role in the immune system by the recognition and removal of antigens, generally bacteria and viruses.

The polymer chain of the antibody is configured to include the so-called heavy and light chains. The basic immunoglobulin unit consists of two identical heavy chains and two identical light chains linked by disulfide bridges. There are five types of heavy chains (α, γ, δ, ε, μ), which determine the immunoglobulin groups (IgA, IgG, IgD, IgE, IgM). The light chain group comprises two subtypes λ and κ.

IgG is a soluble antibody and can be found in blood and other body fluids. It is made by B-cell derived plasma cells in response to bacteria or other pathogens and neutralizes the bacteria or other pathogens. IgG is a Y-shaped glycoprotein with a molecular weight of about 150 kDa and consists of two heavy and two light chains. Each chain is divided into conserved and variable regions. The two carboxy terminal domains of the heavy chain form an Fc fragment (“constant fragment”), and the amino terminal domains of the heavy and light chains recognize the antigen and are referred to as Fab fragments (“antigen-binding fragments”).

Fc fusion proteins are produced through combinations of antibody or Fc fragments with proteins or protein domains that provide specificity for specific drug targets. An example is a domain antibody-Fc fusion protein in which two heavy chains of an Fc fragment are linked to the variable domain (V _H ) of the heavy chain or the variable domain (V _L ) of the light chain. Another Fc fusion protein is a combination of any type of therapeutic protein or protein domain with an Fc fragment. The Fc moiety is contemplated to add stability and portability to protein drugs.

Therapeutic antibodies and Fc fusion proteins are used to treat a variety of diseases, the main examples of which include rheumatoid arthritis, psoriasis, multiple sclerosis and many forms of cancer. The therapeutic antibody may be a monoclonal or polyclonal antibody. Monoclonal antibodies are derived from a single antibody producing cell line, which shows the same specificity for a single antigen. Possible treatments for cancer include antibodies that neutralize tumor cell specific antigens. Bevacizumab (Avastin, Genentech) is a monoclonal antibody that neutralizes vascular endothelial growth factor (VEGF) and prevents neovascular growth into tumor tissue.

Therapeutic fusion proteins such as etanercept (Enbrel, Amgen, TNF-receptor domains linked to Fc fragments) or alefacept (Amevive, Biogen Idec, LFA-3 linked to the Fc portion of human IgG1) may be used for autoimmune diseases. Used or developed as a drug.

Protein bioseparation for the recovery and purification of protein products from various biological feed streams is an important unit operation in the food, pharmaceutical and biotechnology industries. More and more therapeutic monoclonal antibodies (Mabs) and fusion proteins are entering the market or are currently in clinical development. These proteins require very high purity achieved by sophisticated multistep purification protocols. Since downstream processing and refining constitute about 50-80% of the manufacturing cost, considerable efforts are underway to develop new refining strategies or to improve existing refining strategies ( 1 ).

Affinity chromatography is one of the most effective chromatographic methods for protein purification. This is based on highly specific protein-ligand interactions. The ligand is covalently immobilized on the stationary phase used to capture the target protein from the feed stock. Affinity ligands can bind their targets with high specificity and selectivity, allowing higher concentrations up to thousands of times from complex mixtures, resulting in high yields.

Typically, affinity chromatography on Protein A is the first step in most Mab and Fc fusion protein purification schemes. Protein A is a cell wall associated protein exposed to the surface of the bacterium Staphylococcus aureus. It binds with nanomolar affinity to the conserved regions (Fc domains) of various species, in particular human subtypes IgG1, IgG2 and IgG4 ( 2 ). However, the use of Protein A is limited by poor stability under the harsh conditions applied for leaching into the product and sanitary and in situ cleaning procedures. The chemical stability of Protein A can be improved by using genetically engineered Protein A variants for Mab purification. However, efforts have been made to find suitable alternatives, particularly those selected from small molecules, due to the high cost of Protein A resin.

Mabsorbent A2P (Prometic Biosciences) is a small molecule ligand for the purification of immunoglobulins. However, the ligand does not show adequate selectivity ( 3 ) and is not applied in process chromatography.

Another approach uses mixed mode chromatography as the main capture step in antibody purification. The most common substance is based on immobilized 2-mercaptoethylpyridine (MEP HyperCel, Pall Corporation), which effectively captures IgG from fermentation broth but has a lower clearance of host cell protein (HCP) compared to protein A ( 4 ). .

Synthetic affinity ligands that are more readily available, preferably synthetic affinity ligands, which are less expensive than protein-based ligands, are more robust under stringent conditions, have similar or much higher selectivity than protein A, This will provide a suitable solution for antibody and Fc fusion protein purification.

Synthetic small molecule affinity ligands are of particular interest for purification of therapeutic proteins because of their generally higher chemical stability and lower production costs. Synthetic affinity ligands that are more readily available, preferably synthetic affinity ligands, which are less expensive than protein-based ligands, are more vigorous under stringent conditions and have similar or much higher selectivity than protein A, which means that antibodies and Suitable solutions will be provided for Fc fusion protein purification. Depending on the target protein, the affinity ligand should recognize the conserved Fc region of IgG type immunoglobulins and Fc fusion proteins, preferably providing the same broad applicability as protein A.

The underlying problem of the present invention is to provide a matrix comprising an antibody and a small affinity ligand that binds to the Fc region of the Fc fusion protein.

The problem is solved by the embodiments of the present invention described below.

The invention provides a use of a ligand-substituted matrix comprising a support material and at least one ligand covalently bonded to the support material, for affinity purification of the protein, preferably of an antibody or antibody fragment In regards to the use for affinity purification, the ligand is represented by the following formula (I):

Formula (I)

Wherein,

L is the linking point of the support material to which the ligand is attached;

Sp is a spacer group;

v is 0 or 1;

Am is an amide group -NR ¹ -C (O)-, wherein NR ¹ is attached to Ar ¹ and -C (O)-is attached to Ar ² , or -C (O)-is attached to Ar ¹ NR ¹ is attached to Ar ² ;

R ¹ is hydrogen or C ₁ to C ₄ alkyl, preferably hydrogen or methyl, more preferably hydrogen;

Ar ¹ is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring;

Ar ² is (a) a 5- or 6-membered heterocyclic aromatic ring attached to a further 5- or 6-membered aromatic ring via a single bond; Or (b) a 5- or 6-membered heterocyclic aromatic ring fused to an additional 5- or 6-membered aromatic ring as part of a polycyclic ring system; Or (c) C ₁ to C ₄ alkyl, C ₂ to C ₄ alkenyl, C ₂ to C ₄ alkynyl, halogen, C ₁ To C ₄ haloalkyl, hydroxyl-substituted C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, hydroxyl-substituted C ₁ to C ₄ alkoxy, C ₁ to C ₄ alkylamino, C ₁ to C ₄ 5- or 6-membered heterocyclic aromatic ring attached to at least one substituent selected from alkylthio, and combinations thereof.

The ligand-substituted matrix of the invention according to formula (I) has a structure according to formula (Ia) in one embodiment of the invention. In a further embodiment of the invention, the ligand-substituted matrix of the invention according to formula (I) has a structure according to formula (Ib). In formula (la) or (lb), L, Sp, N, R ¹ , Ar ¹ and Ar ² and the integer v have the meanings defined above in relation to formula (I).

Formula (Ia)

Formula (Ib)

Ar ¹ may be represented by AR ¹ , and Ar ² may be represented by AR ² . Sp may also be represented by V. This is the same nomenclature as used in patent application EP 10008089.4, which is claimed as priority in this application. In addition, R ¹ as defined in formula (I) and shown in formulas (Ia) and (Ib) corresponds to R ⁴ as defined and shown in the above priority application.

The ligands according to the invention bind to polyclonal and monoclonal IgG of human origin, in particular human IgG ₁ , IgG ₂ and IgG ₄ and polyclonal and monoclonal IgG of different animal species such as rabbit and mouse immunoglobulins. do.

The present invention further relates to ligands (which may also be referred to as “compounds”) attached to the matrix at the ligand-substituted matrix and at the junction L, optionally via the spacer group Sp.

In formula (I), L is a linking point, which is also referred to as a "point of attachment". Those skilled in the art will identify appropriate connection points / attachments.

It is understood that the connection point L is connected directly to the support material or via a spacer. Typically, L is part of the moiety resulting from the reaction of the appropriate functional group of the support material with the corresponding functional group of the precursor compound of the ligand to form the ligand.

In one embodiment, L is a bond that is attached directly to the support material, generally through a suitable functional group on the support material, preferably a single bond. As used herein, "support material" refers to polymers known and commercially available to those skilled in the art for the purposes of the present invention. The definition of "support material" is given below. In general, the support material comprises a functional group for attachment of a molecule, generally a functional group of the present invention. In this specification, the functional group of the support material is considered as part of the support material; This also applies when the ligand of the invention is linked to the support material via a bond (ie L is a bond and does not comprise a spacer group Sp, see below), wherein said bond is associated with each ligand according to the invention. It is formed directly between the functional groups of the support material.

"Functional group" herein refers to a suitable group on the support material ("precursor group") on the one hand and present on the ligand according to the invention (eg on a C = O group). Or a suitable group (“bulb group”) present on the spacer Sp. In addition, in the present invention, "functional group" denotes an appropriate group ("bulb group") on the spacer group Sp. As used herein, a "precursor group" or "functional group" is a group capable of chemically reacting with a complementary "precursor group" or "functional group" while forming a chemical bond. If necessary, additional reagents may be used for the formation of the chemical bond. As is apparent from the foregoing, during the reaction, the functional group / precursor is transformed into a chemical bond while transforming the original functional group into a "final functionality" or "connecting unit". In the above, it is clear that the functional group / progenitor may also be a "complementary group" or a "complementary group".

Examples of functional groups on the support material are known to those skilled in the art, including -OH, -SH, -NH ₂ ,> NH -COOH, -SO ₃ H, -CHO, -NHNH ₂ , -P (= O) (OH) ₂ , -O-PH (= O) (OH), -P (OH) ₂ , -OP (= O) (OH) ₂ , ester, ether, thioether, epoxide, natural or unnatural amino acid, carboxylic Amides, maleimides, ketones, sulfoxides, sulfones, ureas, isoureas, imidocarbonates, sulfonamides, sulfonylhydrazides, sulfonic esters, phosphates, phosphonates, phosphoramides, phosphonic amides, alkynes, oxime ethers , Oxime, azide, alkoxyamine, semicarbazide, sulfonic halide, phosphonic halide, phosphoric halide, carboxylic active ester, sulfonic active ester, phosphoric active ester, phosphoric active ester, phosphoramidite, Cyanate, thiocyanate, isocyanate, isothiocyanate, maleimide, Vinyl sulfonyl, Cl, Br, I, alkenes, alkyl phosphoniums, including but not limited to.

Examples of functional groups attached to —C═O groups on the ligands of the present invention are known to those skilled in the art, including hydroxy, thiols, F, Cl, Br, esters, ethers, thioethers, carboxylic acids, epoxides, amines, amides, Amino acids, carboxylic amides, maleimides, aldehydes, ketones, sulfonic acids, sulfoxides, sulfones, ureas, isoureas, imidocarbonates, sulfonamides, sulfonic esters, phosphates, phosphonates, phosphoric amides, phosphoric amides, Hydrazine, oxime, azide, alkene, alkyne, hydroxylamine, thiourea, isocyanate, isothiocyanate, N-hydroxysuccinimidyl, 1-hydroxybenzotriazolyl, 7-aza-1-hydroxybenzo Triazolyl, 6-chloro-1-hydroxybenzotriazolyl, including but not limited to.

In this embodiment of the invention, the bond or chemical unit resulting from the reaction of the appropriate functional group on the support material with the appropriate functional group (or “bulb group”) on the —C═O group of the ligand of the invention constitutes the junction L, Wherein the ligand of the invention is attached to the support material / matrix. Thus, the chemical reaction between a functional group on the support material and a functional group on the —C═O group of the ligand of the invention (or “complementary group”) connects the support material and the ligand according to the invention via the junction L.

In a further embodiment of the invention, L is linked to the spacer group -Sp-. In this embodiment, L is a bond or chemical unit resulting from the reaction of a suitable functional group on the support material with a suitable functional group (or “bulb group”) on the spacer group Sp. Thus, the chemical reaction between the functional group on the support material and the functional group (or “complementary group”) on the spacer group Sp connects the support material and the ligand according to the invention via the linking point L.

In the formula (I), when v is 0, the ligand is bound directly to L. In another embodiment, the ligand is bound to the support material via the spacer group Sp. This applies when v is different from 0 in equation (I).

The spacer group Sp is preferably a hydrocarbon group, which may contain further atoms in addition to the C and H atoms. Suitable additional atoms are known to those skilled in the art. Preferred atoms include O, S, N, P, Si. Hydrocarbon groups can be linear or branched or cyclic. Sp is linked to the —C═O group of the ligand according to the invention, and generally Sp is found in the alpha-position relative to the —C═O group. In addition, Sp contains a functional group (a precursor group) to which the ligand of the present invention can be covalently linked to the matrix in a chemical reaction while forming the final functional group (which may also be referred to as a linking unit).

The spacer group Sp preferably contains linking units / final functional groups which covalently link the support material and the ligand via the linking point L. Suitable linking units / final functional groups are known to those skilled in the art. Preferably, the linking unit is -NH ₂ ,> NH, -SH, -COOH, -SO ₃ H, -CHO, -OH, -NHNH ₂ , -P (= O) (OH) ₂ , -O- PH (= O) (OH), -P (OH) ₂ , -OP (= O) (OH) -NHNH ₂ , natural or unnatural amino acids, carboxylic amides, ethers, esters, thioethers, epoxides, Malay Mead, ketone, sulfoxide, sulfone, urea, isourea, imidocarbonate, sulfonamide, sulfonylhydrazide, sulfonic ester, phosphate, phosphonate, phosphoric amide, phosphoric amide, hydrazine, oxime, azide, Alkoxyamines, semicarbazides, sulfonic halides, phosphoric halides, phosphoric halides, carboxylic active esters, sulfonic active esters, phosphoric active esters, phosphoric active esters, phosphoramidites, cyanates, thiocyanates , Alkyl halides, alkyl sulfonates, isocyanates, isothiocyanates, vinyl sulfonyl, carboxyl Derived from suitable precursor groups including lick halide, alkene and alkyne groups.

Typically, the linking unit is attached to a carbon chain into which one or more heteroatoms such as oxygen atoms or carboxamide groups can be inserted. In one embodiment, the linking unit is attached to an alkylene or alkylene-polyoxyalkylene group, such as -CH ₂ -CH _2- (O-CH ₂ -CH ₂ ) x-, wherein x is from 1 to 10 , Preferably it is 1-6. Typically, the functional group on the spacer group is attached to a hydrocarbon group which may contain one or more heteroatoms such as N, O, P, S, Si. Thus, the spacer group is a hydrocarbon group containing at least one heteroatom, preferably a heteroatom selected from N, O, P, S, Si. Preferably, the atoms linked to the C═O groups of the ligands of the invention are nitrogen, oxygen, carbon or sulfur. Suitable chemical entities / chemical units contained in Sp are known to those skilled in the art. Sp generally contains one or more units selected from alkylenes, alcohols, amines, amides, carbonyls, alkyleneoxys and phosphates. The alkylene units preferably have 1-30, preferably 1-12, in particular 1-6 carbon atoms. The alcohol can be primary, secondary or tertiary alcohol. The amines may be inorganic or organic amines. The amines may also be diamines or triamines, preferably diamines. Said amide inorganic or organic amide may be derived from diamines or triamines, preferably diamines. The alkyleneoxy units may comprise linear or branched alkylene units. The alkyleneoxy units are preferably ethyleneoxy or propyleneoxy units, preferably ethyleneoxy units. This unit may also comprise two or more of the aforementioned entities / units, such as amino acids. The amino acid may be a natural or unnatural amino acid. The amino acid may be linear or branched. Branched amino acids can serve as branching points, allowing the linkage of more than one ligand moiety to one point of attachment (“L”). Examples of preferred amino acids include 6-aminohexanoic acid, 8-amino-3,6-dioxaoctanoic acid, 5-amino-3-oxapentanoic acid, glycine, beta-alanine, lysine, ornithine, glutamic acid, 2,3- Diaminopropionic acid, 2,4-diaminobutyric acid and serine.

The spacer group Sp may be composed of one unit described above or may be composed of more than one unit. If the spacer groups Sp consist of more than one unit described above, the units are interconnected by a connecting group. Such linking groups include, but are not limited to, amides, ureas, hydrazides, oxalic diamides, oximes, hydrazones, triazoles, thioureas, isoureas, ethers, and amides and ureas are preferred. Preferably, the phosphate group is provided as a linking group. The phosphate group may be linked to one, two or three units described above. If a phosphate group is linked to the three units described above, it can serve as a branch point, allowing linkage of more than one ligand moiety to one point of attachment (“L”).

Preferred units of the spacer group Sp include amines, preferably organic amines, diamines such as ethylenediamine, piperazine, homopiperazine,-(CH ₂ ) _n -C (= 0), -NH-((CH ₂ ) ₂ O) _n- (CH ₂ ) _n -NH-; -NH- (CH ₂ ) _n -NH-, -NH- (CH ₂ ) _n- , -NH-CH- (C (= 0) NH-, -NH- (OC ₂ H ₄ ) _n -CH _2- C (= 0)-including, but not limited to, where n is an integer from 1-30, preferably from 1-12. In one embodiment, n is an integer from 1-6, i.e. 1, 2 , 3, 4, 5 or 6. Further preferred units of the spacer group Sp include amino acids (which may be natural or unnatural amino acids), in particular 6-aminohexanoic acid, 8-amino-3,6-dioxaoctanoic acid, 5-Amino-3-oxapentanoic acid, lysine and glutamic acid include, but are not limited to, so named units and functional groups are part of the spacer group Sp and part of the junction L.

Particularly preferred examples of the spacer group Sp are -NH- (CH ₂ ) _n NH-, -NH-((CH ₂ ) ₂ O) _n- (CH ₂ ) ₂ -NH-, where n is an integer from 1-30 , Preferably it is an integer of 1-12. In one embodiment n is an integer from 1-6, i.e. 1, 2, 3, 4, 5 or 6.

Particularly preferred examples of the spacer group Sp are also as follows:

-NH-CH ₂ -CH ₂ -CH ₂ -NH, wherein -NH- is attached to the junction L;

-NH- (CH ₂ ) ₃ -NH-, where N is attached to junction L, and the other N is attached to C (= 0) of the ligand;

-N'H-CH (C (= O) NH- (CH ₂ ) ₃ -N''H-) ((CH ₂ ) ₂ C (= O) NH- (CH ₂ ) ₃ -N '''H Where N 'is attached to junction L and N''andN''' are attached to C (= 0) of two separate ligands;

-NH- (CH ₂ ) ₃ -N '(CH ₃ )-, wherein N is attached to the junction L, and N' is attached to the C (= 0) of the ligand;

-N'H- (CH ₂ ) ₃ -NH-C (= O) -CH (-N''H-) (-(CH ₂ ) ₄ -N '''H-), where N' is the junction L N '' and N '''are attached to C (= 0) of two separate ligands;

-N'H- (CH ₂ ) ₃ -NH-C (= O) -CH (-NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -N''H-) (-( CH ₂ ) ₄ -NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -N '''H-), where N' is attached to the junction L, and N '' and N ''' Is attached to C (= 0) of two separate ligands;

-N'H- (CH ₂ ) ₃ -NH-C (= O) -CH (-NH-C (= O) -CH ₂ -OC ₂ H ₄ -N''H-) (-(CH ₂ ) ₄ -NH-C (= 0) -CH ₂ -OC ₂ H ₄ -N '''H-), where N' is attached to the junction L, and N '' and N ''' Attached to C (= 0);

-N'H- (CH ₂ ) ₃ -NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -N''H-, where N 'is attached to the junction L, and N'' Attached to C (= 0) of the ligand;

-N'H- (CH ₂ ) ₃ -NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -N '' H-, where N 'is attached to junction L and N''is attached to C (= 0) of the ligand;

-N'H- (C ₂ H ₄ -O) ₂ -C ₂ H ₄ -NH-C (= O) -NH-C ₂ H _4- (OC ₂ H ₄ ) ₂ -N'H-, where N 'Is attached to the junction L, and the other N' is attached to the C (= 0) of the ligand;

-N'H- (CH ₂ ) ₅ -C (= 0) -NH- (CH ₂ ) ₃ -N''H-, where N 'is attached to the junction L, and N''is C (= Attached to O);

-N'HC ₂ H _4- (OC ₂ H ₄ ) ₂ -NH-C (= O)-(CH ₂ ) ₅ -N''H-, where N 'is attached to the junction L, and N'' Attached to C (= 0) of the ligand;

-N'H- (CH ₂ ) ₆ -NH-C (= O) -CH _2- (OC ₂ H ₄ ) ₂ -N''H-, where N 'is attached to the junction L, and N'' Attached to C (= 0) of ligand.

Typically, the total length of the spacer groups Sp is less than 100 atoms, preferably less than 60 atoms, in particular less than 30 atoms.

In one embodiment of the invention, Sp may be branched. The term "branched" herein means that more than one ligand of the invention according to formula (II) is present on the spacer group Sp. This embodiment allows the connection of more than one ligand to one matrix attachment point (junction point) L.

As used herein, the term "final functionality" or "connecting unit" refers to the reaction between the precursor group on the support material (functional group) and the precursor group on the ligand and / or spacer group Sp of the present invention. The unit formed is shown. The linking unit incorporates a bond between the support material and the ligand of the invention, which may contain a spacer Sp, resulting in a ligand-substituted matrix.

Linking units incorporated into L and / or Sp are known to those skilled in the art and may include the following groups: ethers, thioethers, CC single bonds, alkenyl, alkynyl, esters, amines, amides, carboxylic amides , Sulfonic esters, sulfonic amides, phosphonic esters, phosphonic amides, phosphoric esters, phosphoric amides, phosphoric thioesters, thiophosphoric esters, secondary amines, secondary alcohols, tertiary alcohols, 2-hydroxyamines, Urea, isourea, imidocarbonate, alkylthiosuccinic imide, dialkylsuccinic imide, triazole, ketone, sulfoxide, sulfone, oxime ether, hydrazone, silyl ether, diacyl hydrazide, acyl sulfonyl hydra Zide, N-acyl sulfonamide, hydrazide, N-alkoxy amide, acyl semicarbazide, acyloxy amide, sulfonyloxy amide, phosphoryloxy amide, po Phenyloxy amide, acyl hydrazone, N-carboxamidoisourea, N-carboxamidoimidocarbonate, N-carboxamidoisothiourea, N-carboxamidoimidothiocarbonate, acyl urea, acyl Thiourea, N-carboxamidourea, N-carboxamidoisourea, alkylthioethylsulfonyl, hydrazinylsuccinimide, acylhydrazinylsuccinimide, alkoxyaminosuccinimide, aminosuccinimide, Hydrazinylethylsulfonyl, acylhydrazinylethylsulfonyl, alkoxyaminoethylsulfonyl, aminoethylsulfonyl, tetrazole, alkoxyamidine.

In the formula (I), when v is 0, the ligand is bound directly to L. In another embodiment, the ligand is bound to the support material via the spacer group Sp.

In one embodiment of the invention, R ¹ is selected from: hydrogen; And C ₁ to C ₄ alkyl, typically linear and branched C ₁ to C ₄ alkyl, including cycloalkyl units such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec- Butyl, tert-butyl, cyclopropyl, methylcyclopropyl may be included.

Preferably R ¹ is selected from hydrogen and methyl. More preferably, R ¹ is hydrogen.

Ar ¹ may be an alicyclic or heterocyclic aromatic ring; Preferably Ar ¹ is an alicyclic aromatic ring, ie Ar ¹ is for example phenylene. In a more preferred embodiment, Ar ¹ is phenylene. When Ar ¹ is a heterocyclic aromatic ring, it comprises one, two or more heteroatoms, wherein the heteroatom is preferably selected from N, S, O and combinations thereof. Examples of suitable 5- or 6-membered heterocyclic aromatic rings include pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole ( 4H -1,2,4-triazole and 1H -1,2,4-triazole), 1H -tetrazole, 2H -tetrazole, pyridine, pyrimidine, pyrazine, pyri Chopped, 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine are included, but are not limited to these. Preferred 5- or 6-membered heterocyclic aromatic rings are imidazole, thiazole and pyridine.

Ar ¹ in formula (I) is bonded to a C═O group and an NH group (see formula Ia) or to two C═O groups (see formula Ib).

When represented by formula Ia, in some embodiments the C═O and NH groups are in meta position relative to each other. When Ar ¹ is phenylene, the C═O group and the NH group are positioned meta or para, preferably meta, relative to each other. When the aromatic ring is imidazole, the C═O groups are usually bonded at the 2-position and the NH group is bonded at the 4-position. When the aromatic ring is thiazole, the C═O groups are usually bonded at the 4-position and the NH group is bonded at the 2-position. When the aromatic ring is pyridine, the C═O group and the NH group are preferably located meta relative to each other, while the nitrogen ring atoms may have different positions. In some embodiments, the C═O group is typically bonded at the 3-position of the pyridine ring and the NH group is bonded at the 5-position. Divalent 5- or 6-membered aromatic rings comprising the preferred embodiments described above may be substituted or unsubstituted. Typically, 5- or 6-membered aromatic rings are substituted, preferably monosubstituted. Examples of suitable substituents are C ₁ to C ₄ Alkoxy, typically linear and branched C ₁ to C ₄ Alkoxy (herein cycloalkyl units such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methyl Cyclopropoxy and cyclobutoxy may be included); C ₁ To C ₄ alkyl, typically linear and branched C ₁ to C ₄ alkyl, including cycloalkyl units such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert -Butyl, cyclopropyl, methylcyclopropyl and cyclobutyl); Hydroxyl; And combinations thereof. Preferred substituents include methoxy, ethoxy and methyl, with methoxy being most preferred.

In the most preferred embodiment, Ar ¹ is methoxy-substituted phenylene, wherein the methoxy radical is located in ortho for the C═O group and in para for the NH group (where the C═O and NH groups are For meta). In another preferred embodiment, Ar ¹ is methoxy-substituted phenylene, wherein the methoxy radical is located at para for C═O groups and at ortho for NH groups (where the C═O and NH groups are In the meta for. In another preferred embodiment, Ar ¹ is an N-methyl-substituted imidazole ring, wherein the C═O group is bonded at the 2-position and the NH group is bonded at the 4-position. In another preferred embodiment, Ar ¹ is thiazole, wherein the C═O group is bonded at the 4-position and the NH group is bonded at the 2-position. In another preferred embodiment, Ar ¹ is methyl-substituted phenylene, wherein the methyl radical is located in ortho with respect to the C═O group and in ortho with respect to the NH group (the C═O group and the NH group are in contact with each other). For meta). In another preferred embodiment, Ar ¹ is methyl-substituted phenylene, wherein the methyl radical is located para to the C═O group and to ortho relative to the NH group (the C═O group and NH group relative to each other) In the meta). In another preferred embodiment, Ar ¹ is a pyridine ring, wherein the C═O group is bonded at the 3-position and the NH group is bonded at the 5-position. In another preferred embodiment, Ar ¹ is unsubstituted phenylene, wherein the C═O and NH groups are located meta or para relative to one another.

When represented by formula Ib, in some embodiments two C═O groups are in a meta position relative to each other. When Ar ¹ is phenylene, the two C═O groups are located in meta or para, preferably meta relative to one another.

Ar ² is a 5- or 6-membered unsubstituted or substituted heterocyclic aromatic ring,

(a) attached to an additional 5- or 6-membered aromatic ring via a single bond; or

(b) fused to an additional 5- or 6-membered aromatic ring as part of a polycyclic ring system; or

(c) C ₁ to C ₄ alkyl, C ₂ to C ₄ alkenyl, C ₂ to C ₄ alkynyl, halogen, C ₁ To C ₄ haloalkyl, hydroxyl-substituted C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, hydroxyl-substituted C ₁ to C ₄ alkoxy, C ₁ to C ₄ alkylamino, C ₁ to C ₄ And one or more substituents selected from alkylthio, and combinations thereof.

Ar ² is attached to the C═O group (see formula Ia) or to the NR ¹ group (see formula Ib).

The 5- or 6-membered heterocyclic aromatic ring of Ar ² comprises at least one heteroatom, wherein the heteroatom is preferably selected from N, S, O and combinations thereof. More preferably, the 5- or 6-membered heterocyclic aromatic ring contains two or more nitrogen atoms or nitrogen atoms and oxygen atoms. The C═O group may also be part of the first aromatic ring. Typically, the 5- or 6-membered heterocyclic aromatic ring of Ar ² is attached to the C═O group via a ring heteroatom, preferably a carbon ring atom adjacent to a nitrogen or oxygen atom. In some embodiments, the 5- or 6-membered heterocyclic aromatic ring of Ar ² is attached to the NR ¹ group via a ring heteroatom, preferably a carbon ring atom adjacent to a nitrogen, oxygen or sulfur atom.

Examples of suitable 5- or 6-membered heterocyclic aromatic rings of Ar ² bonded directly to C═O groups or directly to NR ¹ groups in formula (I) include pyrrole, furan, thiophene, imidazole, oxazole, isoxazole , Thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole ( 4H -1,2,4-triazole and 1H -1,2,4-triazole ), 1H -tetrazole, 2H -tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 1 , 2,4 oxadiazole, 1,3,4 oxadiazole, 1,2,4-thiadiazole and 1,3,4-thiadiazole. Preferred 5- or 6-membered heterocyclic aromatic rings are pyrazole, imidazole, isoxazole, pyrrole, furan, 1,2,4-triazole, pyridine, 1,2,4-triazine, pyridazine, pyridine Midine, 1,2,4-oxadiazole, 1,3,4-oxadiazole thiazole and 1,3,4-thiadiazole, most preferably pyrazole, imidazole, isoxazole, 1,2 , 4-oxadiazole, 1,3,4 oxadiazole, thiazole and 1,3,4-thiadiazole. In the case of a pyrazole ring, the C═O group is usually bonded to the pyrazole ring at the 3-position, 4-position or 5-position, preferably the 3-position. In the case of pyrazole rings, the N atoms of the NR ¹ -groups are usually bonded to the pyrazole ring at the 3-position, 4-position or 5-position, preferably the 3-position. In the case of imidazole rings, the C═O groups are usually bonded to the imidazole ring at the 4- or 2-position. In the case of isoxazole rings, the C═O group is typically bonded to the isoxazole ring in the 5-position or 3-position. In the case of a pyrrole ring, the C═O group is typically bonded to the pyrrole ring in the 3-position. In the case of furan rings, the C═O group is typically bonded to the furan ring at the 2-position. In the case of 1,2,4-triazole rings, the C═O groups are usually bonded to the 1,2,4-triazole ring in the 3-position. In a pyridine ring, the C═O group is usually bonded to the pyridine ring in 3-position or 2-position, preferably 2- and 3-position. In the case of 1,2,4-triazine rings, the C═O group is usually bonded to the 1,2,4-triazine ring at the 3-position. In the case of pyridazine rings, the C═O group is usually bonded to the pyridazine ring at the 4-position. In the case of 1,2,4-oxadiazole rings, the C═O group is typically bonded to the 1,2,4-oxadiazole ring at the 5-position. In the case of 1,3,4-oxadiazole rings, the C═O group is typically bonded to the 1,3,4-oxadiazole ring at the 2- or 5-position. In the case of thiazole rings, the N atom of the NR ¹ -group is typically bonded to the thiazole ring at the 2-position. In the case of a 1,3,4 thiadiazole ring, the N atom of the NR ¹ -group is typically bonded to the 1,3,4 thiadiazole ring at the 2-position.

In an alternative (a) of Ar ² described above for Formula (I), the 5- or 6-membered heterocyclic aromatic ring is provided via a single bond an additional 5- or 6-membered aromatic ring (hereinafter referred to as “Ar ² Second aromatic ring ". In these embodiments, the 5- or 6-membered heterocyclic aromatic ring directly bonded to the C═O group (hereinafter referred to as “the first aromatic ring of Ar ² ”) is pyrazole, pyridine, isoxazole, 1,2, Particular preference is given to 4-oxadiazole or 1,3,4-oxadiazole rings. In the case of a pyrazole ring as the first aromatic ring, the C═O group is usually bonded to the pyrazole ring at the 3-position. The second aromatic ring is preferably bonded to the pyrazole ring at the 5-position. In another embodiment, the C═O group is bonded to the pyrazole ring at the 5-position and the second aromatic ring is bonded to the pyrazole ring at the 3-position. When the first aromatic ring is pyridine, the C═O group and the second aromatic ring are preferably located in meta relative to each other, while the nitrogen ring atoms may have different positions. In some embodiments, the C═O group is bonded to the pyridine ring in the 3-position and the second aromatic ring is bonded to the pyridine ring in the 5-position. In another embodiment, the C═O group is bonded to the pyridine ring in the 2-position and the second aromatic ring is bonded to the pyridine ring in the 4-position. In the case of isoxazole rings as the first aromatic ring, the C═O group is usually bonded to the isoxazole ring at the 3-position. The second aromatic ring is preferably bonded to the isoxazole ring at the 5-position. In another embodiment, the C═O group is bonded to the isoxazole ring at the 5-position and the second aromatic ring is bonded to the isoxazole ring at the 3-position. In the case of 1,2,4-oxadiazole rings as the first aromatic ring, the C═O group is usually bonded to the 1,2,4-oxadiazole ring at the 5-position. The second aromatic ring is preferably bonded to the 1,2,4-oxadiazole ring at the 3-position. In the case of a 1,3,4-oxadiazole ring as the first aromatic ring, the C═O group is usually bonded to the 1,3,4-oxadiazole ring at the 2 position. The second aromatic ring is preferably bonded to the 1,3,4-oxadiazole ring at the 5-position. In some embodiments, a 5- or 6-membered heterocyclic aromatic ring (hereinafter referred to as "the first aromatic ring of Ar ² ") directly bonded to the N atom of the NR ¹ -group is pyrazole, thiazole, 1, Particular preference is given to 3,4 thiadiazole rings. In the case of a pyrazole ring as the first aromatic ring, the N atom of the NR ¹ -group is usually bonded to the pyrazole ring at the 5-position. The second aromatic ring is preferably bonded to the pyrazole ring at the 3-position. When the first aromatic ring is a thiazole ring, the N atoms of the NR ¹ -group are typically bonded to the thiazole ring at the 2-position. The second aromatic ring is preferably bonded to the thiazole ring at the 4-position. In another embodiment, the second aromatic ring is preferably bonded to the thiazole ring at the 5-position. In the case of a 1,3,4 thiadiazole ring as the first aromatic ring, the N atom of the NR ¹ -group is usually bonded to the 1,3,4 thiadiazole ring at the 2-position. The second aromatic ring is preferably bonded to the 1,3,4 thiadiazole ring at the 5-position.

In alternative (b), the first aromatic ring is fused to a second 5- or 6-membered aromatic ring as part of a polycyclic ring system, preferably a bicyclic ring system. In the case of pyrazole rings which are part of the fused ring system as the first aromatic ring, the C═O groups are usually bonded to the pyrazole ring at the 3-position. The pyrazole ring is preferably fused to the second aromatic ring via the 4- and 5-positions. In another case, the C═O group is bonded to the pyrazole ring in the 4-position and the second aromatic ring is bonded to the pyrazole ring in the 1- and 5-positions. In another case, the C═O group is bonded to the pyrazole ring in the 3-position and the second aromatic ring is bonded to the pyrazole ring in the 1- and 5-positions. In the case of isoxazole rings which are part of the fused ring system as the first aromatic ring, the C═O group is typically bonded to the isoxazole ring at the 5-position. The isoxazole ring is preferably fused to the second aromatic ring in 3- and 4-positions. In the case of imidazole rings which are part of the fused ring system as the first aromatic ring, the C═O groups are usually bonded to the imidazole ring at the 4-position. The imidazole ring is preferably fused to the second aromatic ring in the 1- and 2-positions. In another case, the C═O group is bonded to the imidazole ring at the 2-position and the second aromatic ring is fused to the imidazole ring via the 4- and 5-positions. In the case of 1,2,4-triazole rings which are part of the fused ring system as the first aromatic ring, the C═O groups are usually bonded to the 1,2,4-triazole ring in the 3-position. The 1,2,4-triazole ring is preferably fused to the second aromatic ring via the 4- and 5-positions. In the case of pyridazine, which is part of the fused ring system as the first aromatic ring, the C═O group is typically bonded to the pyridazine ring at the 4-position. The pyridazine ring is preferably fused to the second aromatic ring via the 5- and 6-positions. For pyrrole rings that are part of a fused ring system as the first aromatic ring, the C═O groups are typically bonded to the pyrrole ring at the 3-position. The pyrrole ring is preferably fused to the second aromatic ring via the 4- and 5-positions. In the case of a pyridine ring which is part of the fused ring system as the first aromatic ring, the C═O group is usually bonded to the pyridine ring at the 2-position. The pyridine ring is preferably fused to the second aromatic ring via the 3- and 4-positions. In the case of 1,2,4-triazine rings which are part of the fused ring system as the first aromatic ring, the C═O groups are usually bonded to the 1,2,4-triazine ring at the 6-position. The 1,2,4-triazine ring is preferably fused to the second aromatic ring via the 3- and 4-positions. In the case of pyrazole rings which are part of the fused ring system as the first aromatic ring, the N atoms of the NR ¹ -groups are usually bonded to the pyrazole ring at the 3-position. The pyrazole ring is preferably fused to the second aromatic ring via the 4- and 5-positions. In the case of imidazole rings which are part of the fused ring system as the first aromatic ring, the N atoms of the NR ¹ -groups are usually bonded to the imidazole ring at the 4-position. The imidazole ring is preferably fused to the second aromatic ring via the 1- and 2-positions. In the case of a thiazole ring which is part of a fused ring system as the first aromatic ring, the N atom of the NR ¹ -group is usually bonded to the thiazole ring at the 2-position. The thiazole ring is preferably fused to the second aromatic ring via the 4- and 5-positions.

In two alternatives (a) and (b) comprising the preferred embodiments described above, the second aromatic ring of Ar ² is typically a 5- or 6-membered alicyclic or heterocyclic ring. Examples of suitable 5- or 6-membered aromatic rings include benzene, pyrrole, furan, thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, 1,2,3-triazole, 1 , 2,4-triazole ( 4H -1,2,4-triazole and 1H -1,2,4-triazole), 1H -tetrazole, 2H -tetrazole, pyridine, pyrimidine, pyrazine, pyridazine , 1,2,3-triazine, 1,2,4-triazine and 1,3,5-triazine, but are not limited to these. Preferred second aromatic rings are benzene, furan, thiophene, pyrrole, pyridine, thiazole, pyrimidine and pyrazole, most preferably benzene, thiazole, furan and thiophene.

In alternative (a), the second aromatic ring of Ar ² is typically furan, thiophene, pyrrole, benzene, pyridine or thiazole, preferably furan, thiophene, benzene and thiazole, most preferably furan and thia boil down. In the case of furan, thiophene, pyrrole, benzene or thiazole rings as the second aromatic ring in alternative (a), the rings are preferably bonded to the first aromatic ring via the 2-, 3- or 4-position, Most preferred are the 2- and 4-positions. In a typical embodiment of alternative (a), the first aromatic ring is a pyrazole ring and the second aromatic ring is a furan ring, ie Ar ² is furylpyrazyl, such as 5- (2-furyl) pyrazol-3-yl to be. In further exemplary embodiments, Ar ² is 5- (2-methylthiazol-4-yl) isoxazol-3-yl, 4- (2-furyl) pyridin-2-yl, 3- (4-methoxyphenyl )-[1,2,4] oxadiazol-5-yl, 3- (2-furyl)-[1,2,4] oxadiazol-5-yl or 5- (2-furyl)-[1 , 3,4] oxadiazol-2-yl.

In alternative (b), the second aromatic ring of Ar ² is preferably benzene, thiazole, pyrrole, pyrimidine or pyridine, most preferably benzene and thiazole. In typical embodiments of alternative (b), Ar ² is benzo [c] isoxazol-3-yl, imidazo [2,1-b] thiazol-6-yl, cynolin-4-yl, indole-3 -yl, indazol-3-yl, benzo [c] furan-1-yl, 1-isoquinolinyl, 5-oxo -5 H - [1,3] thiazol [3,2-a] pyrimidine -6-yl, pyrazolo [1,5-a] pyrimidin-3-yl, [1,2,4] triazolo [4,3-a] pyrimidin-3-yl and pyrazolo [5,1 -c] [1,2,4] triazin-3-yl, preferably benzo [c] isoxazol-3-yl, indazol-3-yl and imidazo [2,1-b] thiazole -6- days.

In alternative (c) of Ar ² described above for formula (I), the 5- or 6-membered heterocyclic aromatic ring is C ₁ to C ₄ alkyl, typically linear or branched C ₁ to C ₄ alkyl (which is Cycloalkyl units such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl, cyclopropyl, methylcyclopropyl and cyclobutyl); C ₂ to C ₄ alkenyl; C ₂ to C ₄ alkynyl, such as ethynyl; Halogen such as fluoro, chloro, bromo and iodine; C ₁ To C ₄ haloalkyl, such as trifluoromethyl; Hydroxyl-substituted C ₁ to C ₄ alkyl, typically mono- or dihydroxyalkyl, such as mono- or dihydroxyethyl or mono- or dihydroxypropyl; C ₁ to C ₄ alkoxy, typically linear and branched C ₁ To C ₄ Alkoxy (which is a cycloalkyl unit such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy, cyclopropoxy, methylcyclo Propoxy and cyclobutoxy may be included); Hydroxyl-substituted C ₁ to C ₄ alkoxy, typically monohydroxyl-substituted C ₁ to C ₄ alkoxy; C ₁ to C ₄ alkylamino; Attached to one or more (preferably one or two) substituents selected from C ₁ to C ₄ alkylthio, and combinations thereof. The substituent (s) may be bonded to the ring via ring carbon and / or ring heteroatoms, preferably via carbon and / or nitrogen ring atoms. Preferred substituents of the 5- or 6-membered heterocyclic aromatic ring in alternative (c) are cyclopropyl, ethynyl, methyl and trifluoromethyl. One or more substituents are preferably located in the meta relative to the C═O group. In typical embodiments of alternative (c), Ar ² is 3-cyclopropylpyridin-5-yl, 3-ethynylpyridin-5-yl, 5- (trifluoromethyl) pyrazol-3-yl, 5- (Trifluoromethyl) pyrazol-3-yl or 1-methyl-5- (trifluoromethyl) pyrazol-3-yl.

In alternatives (a) and (b) the aromatic rings of Ar ² , ie, the first aromatic ring or the second aromatic ring or both, are typically optionally substituted with one or more further substituents selected from the substituents described above for alternative (c). It is understood that it may contain. The substituent (s) may be bonded to the ring (s) via ring carbon or ring heteroatoms, preferably carbon or nitrogen atoms. Preferred substituents of the first and / or second aromatic rings of Ar ² are C ₁ to C ₄ alkyl, such as methyl; Halo such as bromo and chloro; Hydroxyalkyl such as methoxy, 2-hydroxyethyl or 2,3-dihydroxypropyl, alkylamino such as aminopropyl and haloalkyl such as trifluoromethyl. When the first aromatic ring is a pyrazole ring, it is usually methyl-substituted, preferably N-methyl-substituted and more preferably substituted at the 1-position. N-methyl substitution of the pyrazoles is particularly preferred in alternative (a).

A typical example of Ar ² substituted in alternative (a) is 5- (2-furyl) -1-methylpyrazol-3-yl.

If the first aromatic ring is a pyridine ring, it usually does not contain further substituents.

Typical examples of Ar ² substituted in alternative (b) are 5-chloro-1 H -indazol-3-yl, 1-methylindol-3-yl, 1-methylindazol-3-yl, 5,7- Dimethyl- [1,2,4] triazolo [4,3-a] pyrimidin-3-yl 4,7-dimethylpyrazolo [5,1-c] [1,2,4] triazine-3- 1-ethylindazol-3-yl, 1- (2-hydroxyethyl) indazol-3-yl and 1- (2,3-dihydroxypropyl) indazol-3-yl.

It is also included in the present invention that in two alternatives (a) and (b) the second aromatic ring can optionally be fused to an additional aromatic ring such as benzene. A typical example of a second aromatic ring fused to an additional aromatic ring is benzothiophene.

In a preferred embodiment of the invention, the group in alpha position relative to the carbonyl functional group is the amino group NR ² , wherein R ² may have various meanings as defined below. In this case, the ligand-substituted matrix of the present invention can be represented as in the following formulas (II), (IIa) and (IIb) covered by the above formulas (I), (Ia) and (Ib).

[Formula (II)

Formula (IIa)

Formula (IIb)

In the formulas (II), (IIa) and (IIb), the NR ² group is not shown as part of the spacer group Sp or junction L, but as part of the ligand for reasons of clarity. However, it should be noted that NR ² together with V form a spacer group Sp, as shown in formulas (I), (Ia) and (Ib), or are part of the junction L.

In one embodiment of the invention, R ² is selected from: hydrogen; And C ₁ to C ₄ alkyl, typically linear and branched C ₁ to C ₄ alkyl, which are cycloalkyl units such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl , tert-butyl, cyclopropyl, methylcyclopropyl).

Preferably R ² is selected from hydrogen and methyl. More preferably, R ¹ is hydrogen.

The other symbols Ar ¹ , Ar ² and Am have the meanings and preferred meanings defined above for the formulas (I), (la) and (lb).

R ² defined in formula (II) and shown in formulas (IIa) and (IIb) corresponds to R ³ defined and shown in patent application EP 10008089.4, which is claimed as priority in the present application.

The invention also encompasses the ligand of the invention (after being attached to the support material) together with the support material to form the ligand-substituted matrix used in the methods of the invention. The ligand is according to the following formula, wherein the symbols Sp, Ar ¹ , Ar ² and Am have the meanings defined above including the preferred ones.

[Formula (III)]

Formula (IIIa)

Formula (IIIb)

Ligands according to formulas (III), (IIIa) and (IIIb) comprise a spacer group Sp attached to the ligand.

The ligands according to formulas (III), (IIIa) and (IIIb) can serve as precursors for the synthesis of further compounds to which no spacer groups are attached. The compounds named above are prepared after cleavage of the spacer group and optionally after conversion of —C═O comprising a functional group present in the portion of the molecule that binds the antibody to any other suitable functional group known to those skilled in the art. do. Suitable reactions for this purpose are known to those skilled in the art. The resulting compounds are also included in the present invention.

Preferred ligands of the matrix according to the invention are shown below, in which the leftmost N atom in each formula is connected to a junction L not shown. All of the following formulas show spacer groups attached to the ligand:

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L1)

5- [5- (4-Bromo-2-thienyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L2)

5- [5- (2,5-dimethyl-3-thienyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L3)

5- (5-Chloro-1 H -indazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L4)

2-methoxy-5- [3- (2-thienyl) pyridin-5-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L5)

2-methoxy-5- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L6)

5- [5- (3-Chlorophenyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L7)

2-methoxy-5- [1-methyl-5- (3-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L8)

2-Methyl-3- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L9)

4-Methyl-3- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L10)

2-methoxy-5- (3-phenylpyridin-5-yl) carboxamidobenzoic acid N- (3-aminopropyl) amide (L11)

2-methoxy-5- {3- [4- (trifluoromethyl) phenyl] pyridin-5-yl} carboxamidobenzoic acid N- (3-aminopropyl) amide (L12)

5- [3- (2-furyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L13)

5- [3- (2-benzothienyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L14)

2-methoxy-5- (1-methyl-5-phenylpyrazol-3-yl) carboxamidobenzoic acid N- (3-aminopropyl) amide (L15)

2-methoxy-5- [5- (1-methyl-2-pyrrolyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L16)

3- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L17)

4- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L18)

2- [1-Methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidothiazole-4-carboxylic acid N- (3-aminopropyl) amide (L19)

5- [5- (2-furyl) pyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L20)

3- { N- [5- (2-furyl) pyrazol-3-yl] carbamoyl} benzoic acid N'- (3-aminopropyl) amide (L21)

5- [3- (3-furyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L22)

5- (3-Cyclopropylpyridin-5-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L23)

5- (3-ethynylpyridin-5-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L24)

2-methoxy-5- (1-methylindol-3-yl) carboxamidobenzoic acid N- (3-aminopropyl) amide (L25)

5- (Indol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L26)

2-methoxy-5- (1-methylindazol-3-yl) carboxamidobenzoic acid N- (3-aminopropyl) amide (L27)

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) -N -methylamide (L28)

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-ethoxybenzoic acid N- (3-aminopropyl) amide (L29)

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-hydroxybenzoic acid N- (3-aminopropyl) amide (L30)

5- (Benzo [c] isoxazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L31)

5- (benzo [c] furan-1-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L32)

5- (1,5-dimethylpyrazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L33)

2-methoxy-5- [1-methyl-5- (trifluoromethyl) pyrazol-3-yl] carboxamidobenzoic acid N- (3-aminopropyl) amide (L34)

5- (1-Isoquinolinyl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L35)

5- (indazol-3-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L36)

5- (imidazo [2,1-b] thiazol-6-yl) carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L37)

5- (1-ethylindazol-3-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L38)

5- [ N- (1-methylindazol-3-yl) carbamoyl] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L39)

( RS ) -5- [1- (2,3-dihydroxy-1-propyl) indazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L40 )

5- ( 3H -imidazo [4,5-b] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L41)

2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido] benzoic acid (3-amino-1-propyl) amide (L42)

2-methoxy-5-([1,2,4] triazolo [4,3-a] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L43)

2-methoxy-5- (1-methylpyrazolo [3,4-b] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L44)

5- [5- (2-furyl) isoxazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L45)

2-methoxy-5- (pyrazolo [1,5-a] pyridin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L46)

5- ( 1H -benzimidazol-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L47)

5- (imidazo [1,2-b] pyridazin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L48)

( S ) -2,6-bis [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] hexanoic acid (3-amino- 1-propyl) amide (L49)

( S ) -2,6-bis {8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6 Dioxaoctanoyl amido} hexanoic acid (3-amino-1-propyl) amide (L50)

2-methoxy-5- (3-phenylisoxazole-5-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L51)

5- [4- (2-furyl) pyridin-2-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L52)

( S ) -2,6-bis {5- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3-oxa Pentanoyl amido} hexanoic acid (3-amino-1-propyl) amide (L53)

2-methoxy-5- [3- (4-methoxyphenyl) -1,2,4-oxadiazole-5-ylcarboxamido] benzoic acid (3-amino-1-propyl) amide (L54)

5- [3- (2-furyl) -1,2,4-oxadiazole-5-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L55)

2-methoxy-5- (pyrazolo [1,5-a] pyrimidin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L56)

5- (imidazo [1,2-a] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L57)

5- [5- (2-furyl) -1,3,4-oxadiazol-2-yl] carboxamido-2-methoxybenzoic acid (3-amino-1-propyl) amide (L58)

5- [1- (2-hydroxyethyl) indazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L59)

8- [2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamido] -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L60 )

8- {8- [2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamido] -3,6-dioxaoctanoylamido} -3,6- Dioxaoctanoic acid (3-amino-1-propyl) amide (L61)

( S ) -2,6-bis {8- [5- (imidazo [1,2-b] pyridazine-2-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6 Dioxaoctanoyl amido} hexanoic acid (3-amino-1-propyl) amide (L62)

5- (imidazo [1,2-a] pyrimidin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L63)

( S ) -1-Aminopropane-1,3-dicarboxylic acid bis {3- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarbox Amido] propyl} amide (L64)

8- {8- {5- [5- (2-furyl) -1-methylpyrazol-3-ylcarboxamido] -2-methoxyphenylcarboxamido} -3,6-dioxaoctanoyl Amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L65)

8- {8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoylamido } -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L66)

8- {8- {5- [5- (2-furyl) -1,3,4-oxadiazole-2-ylcarboxamido] -2-methoxyphenylcarboxamido} -3,6- Dioxaoctanoyl amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L67)

8- {8- {2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido] phenylcarboxamido} -3,6-dioxa Octanoyl amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L68)

N- (8-amino-3,6-dioxaoctyl) -N ' -{8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxy Phenylcarboxamido] -3,6-dioxaoctyl} urea (L69)

Isophthalic acid N- (3-aminopropyl) -N'- [5- (2-furyl) -1,3,4-thiadiazol-2-yl] amide (L70)

Isophthalic acid N - (3- aminopropyl) - N'- (1- methyl-indazol-3-yl) amide (L71)

Isophthalic acid N- (3-aminopropyl) -N'- (benzothiazol-2-yl) amide (L72)

Isophthalic acid N- (3-aminopropyl) -N'- (4-phenylthiazol-2-yl) amide (L73)

Isophthalic acid N- (3-aminopropyl) -N'- (5-phenylthiazol-2-yl) amide (L74)

5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxybenzoic acid [3- (6-aminohexanoylamido) -1-propyl] amide (L75)

6- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] hexanoic acid (8-amino-3,6-dioxa- 1-octyl) amide (L76)

8- [5- (imidazo- [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoic acid (3-amino- 1-propyl) amide (L77)

8- [5- (imidazo- [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoic acid (6-amino- 1-hexyl) amide (L78)

The synthesis of ligand (II) can be carried out, for example, on an insoluble support (which is also known as a resin, such as a polystyrene resin), and is preferably reactive with spacer groups such as Fmoc protected amino acids that can be attached by amide formation. It may be carried out on an insoluble support pre-loaded with the desired linker containing groups such as amino groups. After deprotection of the spacer groups, the remaining portion of the molecule is attached by any reaction that results in the formation of appropriate bonds, if necessary by further synthetic steps (such as nucleophilic transfer reactions). Finally, ligands containing a spacer group (linker moiety) are released from the insoluble support / resin by appropriate cleavage protocols known to those skilled in the art and purified by chromatography methods also known to those skilled in the art.

If a suitable solid-phase synthesis protocol should not be applicable to a particular ligand, the synthesis can optionally be carried out in solution. Typically, the solution phase synthesis consists of the synthesis steps necessary for the conjugation reaction (eg, amide formation) between the ligand precursor and the suitable spacer group and the assembly of the ligand itself. In some cases, protecting groups are required to prevent side effects.

The term "antibody" refers to an immunoglobulin, which includes natural or wholly or partially synthetically prepared, and includes all fragments and derivatives thereof that retain specific binding capacity. Typical fragments are Fc, Fab, heavy and light chains. The term also includes any polypeptide having a binding domain that is homologous or highly homologous to an immunoglobulin binding domain (such as having at least 95% identity when comparing amino acid sequences). Such polypeptides may be derived from natural sources or may be synthetically prepared in part or in whole. The antibody may be monoclonal or polyclonal, may be of human or nonhuman origin, or the human Fc moiety is fused to a murine Fab fragment (therapeutic antibodies such as rituximab) ending in ximab or human and murine sequences It may be a chimeric protein fused to a Fab fragment comprising a therapeutic antibody ending in zumab (such as bevacizumab) or linked to different Fab fragments (bispecific antibodies). The antibody may be a member of any group of immunoglobulins, preferably IgG, more preferably IgG ₁ , IgG ₂ and IgG _{4 in} the case of human antibodies.

In the most preferred embodiment of the invention, the antibody comprises an Fc fragment or domain, because the ligand according to the invention is believed to bind to the Fc portion of the antibody. Thus, the antibodies of the invention are most preferably Fc of the immunoglobulin group, preferably IgG, more preferably human IgG or polyclonal or monoclonal IgG of human origin, in particular IgG ₁ , IgG ₂ and IgG ₄ Antibodies containing fragments or domains.

The term “Fc fusion protein” refers to any combination of one or more proteins or protein domains with an Fc fragment. Examples of Fc fusion proteins include chimeric (therapeutic proteins ending in cept) of the soluble receptor domain and the human IgG ₁ Fc domain, such as etanercept (human IgG ₁ Fc with two tumor necrosis factor receptor domains combined) or lilona. Septs (human IgG ₁ fused with interleukin-1-receptor domains) are included, but are not limited to these. Fc fusion proteins can be prepared by any means. For example, an Fc fusion protein can be prepared chemically or enzymatically by coupling an Fc fragment to an appropriate protein or protein domain, or can be recombinantly produced from a gene encoding the Fc and protein / protein domain sequences. Alternatively, the Fc fusion protein may be synthetically prepared in whole or in part. Fc fusion proteins may also optionally be multimolecular complexes. Functional Fc fusion proteins will typically comprise at least about 50 amino acids, more typically at least about 200 amino acids.

The present invention relates to affinity purification of antibodies or Fc fusion proteins from complex mixtures such as fermentation supernatants or plasma of human or animal origin, preferably by affinity chromatography, using affinity ligands of formula (I). And to preferred embodiments thereof, as disclosed elsewhere herein.

Thus, in a preferred embodiment, the invention comprises a method for purifying a protein, preferably an immunoglobulin or an Fc fusion protein, by affinity purification, preferably affinity chromatography. Affinity ligands according to the invention bind to the Fc region of an antibody.

Bevacizumab (Avastin®, Genentech / Roche ) is a humanized monoclonal antibody. It prevents neovascularization by targeting and inhibits tumor growth by inhibiting the function of protein vascular endothelial growth factor-A (VEGF-A), which stimulates angiogenesis .

Tocilizumab (RoActemra®, Genentech / Roche ) is a humanized monoclonal antibody. It is against the interleukin-6 receptor and blocks the action of proinflammatory cytokine interleukin-6. It is approved for the treatment of rheumatoid arthritis.

Palivizumab (Synagis®, Abbott) is a humanized monoclonal antibody against the A-epitope of Respiratory Syndrome Virus (RSV) F-protein. It is used to prevent RSV infection.

Polyclonal human and rabbit IgGs were investigated to demonstrate the general applicability of the disclosed ligands for affinity purification of immunoglobulins via the Fc moiety.

In the practice of the present invention, the ligand according to formula (I) is attached to a support matrix of a suitable support material, whereby a ligand-substituted matrix for protein separation, usually affinity purification, preferably affinity chromatography A matrix for graphics is created, which is also referred to herein as an affinity matrix. The ligand of the general formula is attached to the support matrix via L, optionally comprising a spacer -Sp-.

Accordingly, the present invention includes a support material and one or more ligands as previously described herein, wherein the ligands comprise a ligand-substituted matrix (affinity matrix) for protein separation, attached via L.

The support matrix (which may be designated M) may comprise any suitable support material known to those skilled in the art. Such materials may be soluble or insoluble, particulate or nonparticulate, or monolithic structures, including fibers and membranes, porous or nonporous. This provides conventional means for separating the ligands of the present invention from the solute in the contact solution. Examples of support matrices include carbohydrates and crosslinked carbohydrate matrices such as agarose, sepharose, sefadex, cellulose, dextran, starch, alginate or carrageenan; Synthetic polymer matrices such as polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG polyacrylate copolymers, polymethacrylates such as poly (hydroxyethyl methacrylate), polyvinyl alcohol, polyamides or purple Luorocarbon; Inorganic matrices such as glass, silica or metal oxides; And combinations thereof.

Affinity matrices are prepared by providing a support matrix of a suitable support material and attaching a ligand of formula (I) thereto. Methods of attaching ligand (I) to a support material are known to those skilled in the art.

The invention also relates to a method for affinity purification, preferably affinity chromatography, of a protein, wherein the protein to be purified is contacted with a ligand-substituted matrix as described above.

The term “affinity purification” (used interchangeably with the term “affinity separation”) refers to any separation technique involving molecular recognition of a protein by a ligand of formula (I). The ligand may later be immobilized on a solid support that facilitates the separation of the ligand-antibody complex. Separation techniques may include, but are not limited to, affinity chromatography on packed columns, monolithic structures, or membranes. The term also includes batch-type adsorption or affinity precipitation.

Independent of the flavor, the purification technique is constructed by the initial recognition period when the ligand is in contact with the crude antibody. In the second phase, impurities are separated from ligand-antibody complexes (eg column chromatography) or ligand-antibody complexes are separated from impurities (eg affinity precipitation). In a third step, the antibody is ligand-antibody by modification of chemical and / or physical conditions such as changes in pH, ionic strength, and / or by the addition of a modifier such as an organic solvent, detergent or chaotrope. Is released from the complex.

The invention will be illustrated by the following examples, which should not be considered as limiting the invention.

The documents cited herein:

1.A.Cecilia, A.Roque, C.R. Lowe, M.A. Taipa: Antibodies and Genetically Engineered Related Molecules: Production and Purification, Biotechnol. Prog. 2004, 20, 639-654

2. K. L. Carson: Flexibility-the guiding principle for antibody manufacturing, Nature Biotechnology, 2005, 23, 1054-1058; S. Hober, K. Nord, M. Linhult: Protein A chromatography for antibody purification, J. Chromatogr. B. 2007, 848, 40-47

3.T.Arakawa, Y.Kita, H.Sato, D. Ejima, Protein Expression and Purification. 2009, 63, 158-163

4. S. Ghose, B. Hubbard, S.M. Cramer, Journal of Chromatography A, 2006, 1122, 144-152

Materials and methods

Unless otherwise stated, all chemicals and solvents are of analytical grade except Example 2. The reagents used in Example 2 are preliminary for analytical grade depending on particle requirements and availability.

96 and 384-well filter plates with hydrophilic membrane filters with an average pore size of 0.45 μm were purchased from Pall GmbH (Dreieich / Germany). Top frit made from polyethylene with an average pore size of 10 μm was provided by Porex (Bautzen / Germany). General purpose microtiterplates for fraction collection and analysis assays were ordered from Greiner Bio One GmbH (Frickenhausen / Germany). Assay assays were read using a Fluostar Galaxy plate reader from BMG Labtech GmbH (Offenburg / Germany).

Column chromatography of antibodies and purification of antibody fragments were performed on a Waters HPLC system (Waters GmbH, Eschborn / Germany). Omnifit column housings (Diba Industries Ltd, Cambridge / United Kingdom) were used for packing the columns. NHS-activated Sepharose 4 FF, rProtein A Sepharose FF and Superdex 70 chromatography media were purchased from GE Healthcare (Uppsala / Sweden). Mavsorvent A2P HF was purchased from Prometic Life Sciences (Cambridge / United Kingdom) and MEP Hypercel was purchased from Pall Corporation (Port Washington NY, USA).

Analytical chromatography of the ligand was performed on a Shimadzu HPLC system (Shimadzu Deutschland GmbH, Duisburg / Germany), including a diode array detector and a single-quad mass spectrometer. Monolithic C18 reversed phase columns were purchased from Merck KGaA (Darmstadt / Germany). The solvent used for analysis was mass spectrometer grade.

Antibodies used in the present invention are bevacizumab (Avastin, F. Hoffmann-La Roche, Switzerland), tocilizumab (RoActemra, F. Hoffmann-La Roche, Switzerland), Palivizumab (Synagis, Abbott, USA) , Poly-IgG from human serum (Sigma-Aldrich, USA) and poly-IgG from rabbit serum (Sigma-Aldrich, USA). Immobilized papain for preparing antibody fragments was purchased from Thermo Scientific (Bonn / Germany).

Flowthrough of Protein A chromatography (referred to as host cell protein) was derived from the supernatant of antibody-producing CHO cell culture in serum free medium.

Coomassie Brilliant Blue Dye Reagent for Bradford analysis was purchased from Thermo Scientific (Bonn / Germany).

Example 1

SPR Screening of Chemical Microarrays

Graffinity has developed a high-density chemical microarray containing thousands of small molecules immobilized on gold chips. The array is constructed using maleimide-thiol coupling chemistries in combination with high density pintool spotting. For this configuration, the glass plate is microstructured by the photolithographic method to define up to 9,216 sensor fields per array. The gold coating then applied provides the basis for the SPR effect and allows the formation of a binary mixed self-assembled monolayer (SAM) of two different thiols. One of the thiols represents a reactive maleimide moiety to which the thiol-tagged array compound can couple during pintool spotting. The resulting microarray contains 9,216 sensor fields, each containing multiple copies of a defined and quality controlled array compound.

Surface plasmons are collective oscillations of electrons at the metal surface that can be resonated and excited by polarization of the appropriate wavelength and angle of incidence. Under surface plasmon resonance conditions, the intensity of light reflected from the gold chip shows a sharp attenuation (SPR minimum). The angle and wavelength position of this reflectivity minimum depends largely on the refractive index of the medium adjacent the metal surface. Thus, processes that change the local refractive index in close proximity to the metal surface (eg, antibodies that bind to immobilized ligands) can be sensitively monitored. For SPR detection, chemical microarrays are incubated with the target protein under optimized screening conditions. If the target binds to the immobilized fragment, a shift in wavelength dependent SPR minimum (referred to as SPR shift) occurs with respect to the buffer control and thus represents protein-ligand interaction. The SPR imaging approach uses an extended beam of parallel light that illuminates the entire microarray sensor region at a fixed angle. The reflected light is then captured by the CCD camera. Reflected images are recorded in stages, scanning over various wavelengths covering SPR resonance conditions. The automated spot discovery routine and subsequent gray-scale analysis of the acquired photographs simultaneously generate SPR resonance curves of all 9,216 sensor fields per chip.

Screening of Antibodies and Fragments thereof by SPR

The platform allows for the screening of proteins, antibodies and fragments thereof against a fixed compound library of 110,000 small molecules (Graffinity) in a high-throughout fashion. For each target, SPR screening conditions were individually optimized prior to screening of targets for the entire library. Among such optimized screening parameters are a) composition of the screening buffer comprising appropriate detergent, b) concentration of antibody or protein target in solution, and c) surface density of immobilized ligand on the chip surface. Neumann et al. Summarize the screening targets applied and the corresponding screening conditions. In total, four different full length antibodies were screened and compared. In addition, Fc and Fab fragments of two antibodies were obtained by proteolytic digestion, and array screening was also performed. To identify antibody specific ligands, the screening activity also included non-antibody host cell proteins (HCPs) of CHO cell lines as controls.

Experimental results of microarray screening activities were analyzed not only by manual hit selection using in-house developed software routines, but also by in-depth data analysis by commercial data mining tools. It became. Many hit series derived by this analysis showed clear binding to the antibody and its Fc fragments, but only weakly negligible binding to the HCP control. This primary hit series was used for hit identification in secondary assays and could be used as a starting point for the synthesis of libraries concentrated around the primary hit. For example, the Fc specific ligand L1 showed clear binding to full length bevacizumab and its Fc fragments, but only minor binding to the bevacizumab Fab fragment. Moreover, it clearly bound other full length antibodies anti RhD IgG, tocilizumab and palivizumab.

Example 2

Synthesis of Ligands

General Method A: Synthesis of 5-arylcarboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide (L1, L2, L3, L6, L8, L7, L15, L16, L5, L11, L20 , L27, L25, L4, L26, L31, L32, L33, L34, L35, L36, L42, L44, L45, L46, L47, L48, L51, L56, L57, L63)

N- Fmoc-5-amino-2-methoxybenzoic acid (0.2-0.5 mmol) and HOAt (1 equivalent to carboxylic acid) are dissolved in DMF, NMP, DMF / DMSO or NMP / DMSO mixture (1-3 mL) , DIC (1 equivalent to carboxylic acid). After stirring for 2-5 minutes, the mixture was added to 0.1-0.15 mmol of 1,3-diaminopropane linked to trityl-polystyrene resin or 2-chlorotrityl polystyrene resin. The mixture was shaken for several hours or overnight. Next, the resin was washed (DMF or DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times) and treated with 25% piperidine in DMF (1-5 mL) for 15-30 minutes. It was. The resin was then washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times) and dried in air, in a nitrogen stream, or in high vacuum.

Arylcarboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent to carboxylic acid) are dissolved in DMF, NMP, DMF / DMSO or NMP / DMSO mixture (1-3 mL) and treated with DIC (1 equivalent to carboxylic acid) It was. After stirring for 2-5 minutes, the mixture was added to the resin and shaken for several hours or overnight. After washing (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), the target compound was subjected to a suitable cleavage mixture (dichloromethane, TFA, triethylsilane, 85: for trityl resin). 10: 5, 45:45:10) for 2-chlorotrityl resin) to decompose from the support. Typically, the decomposition step was repeated once, followed by rinsing the resin with dichloromethane. After evaporation of the solvent, the crude residue was purified by preparative reverse phase HPLC.

With regard to the high robustness of the amide coupling chemistry, other coupling protocols can be applied with similar or identical results. In particular, TBTU or HATU coupling (1 equivalent for carboxylic acid; additionally 2 equivalents of DIPEA must be added) represents a reliable alternative to faster DIC / HOAt coupling (typical reaction time: 30 minutes to 3 hours); However, for some carboxylic acids, especially for carboxylic acids containing free aryl NH, such as indole or (benzo) pyrazole, coupling under basic conditions can result in unwanted byproducts.

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L1)

Prepared from 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 398 (M + 1).

5- [5- (4-Bromo-2-thienyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L2)

Prepared from 5- (4-bromo-2-thienyl) -1-methylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 493, 495 (M + l).

5- [5- (2,5-dimethyl-3-thienyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L3)

Prepared from 5- (2,5-dimethyl-3-thienyl) -1-methylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 442 (M + l).

2-methoxy-5- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L6)

Prepared from 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid according to general method A. ESI-MS: 414 (M + l).

2-methoxy-5- [1-methyl-5- (3-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L8)

Prepared from 1-methyl-5- (3-thienyl) pyrazole-3-carboxylic acid according to general method A. ESI-MS: 414 (M + l).

5- [5- (3-chlorophenyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L7)

Prepared from 5- (3-chlorophenyl) -1-methylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 442, 444 (M + l).

2-methoxy-5- (1-methyl-5-phenylpyrazol-3-yl) carboxamidobenzoic acid N -(3-aminopropyl) amide (L15)

Prepared from 1-methyl-5-phenylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 408 (M + l).

2-methoxy-5- [5- (1-methyl-2-pyrrolyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L16)

Prepared from 5- (1-methyl-2-pyrrolyl) -1-methylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 411 (M + 1).

2-methoxy-5- [3- (2-thienyl) pyridin-5-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L5)

Prepared from 3- (2-thienyl) pyridine-5-carboxylic acid according to general method A. ESI-MS: 411 (M + 1).

2-methoxy-5- (3-phenylpyridin-5-yl) carboxamidobenzoic acid N -(3-aminopropyl) amide (L11)

Prepared from 3-phenylpyridine-5-carboxylic acid according to general method A. ESI-MS: 405 (M + l).

5- [5- (2-furyl) pyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L20)

Prepared from 5- (2-furyl) pyrazole-3-carboxylic acid according to general method A. ESI-MS: 384 (M + 1).

2-methoxy-5- (1-methylindazol-3-yl) carboxamidobenzoic acid N -(3-aminopropyl) amide (L27)

Prepared from 1-methylindazole-3-carboxylic acid according to general method A. ESI-MS: 382 (M + 1).

2-methoxy-5- (1-methylindol-3-yl) carboxamidobenzoic acid N -(3-aminopropyl) amide (L25)

Prepared from 1-methylindole-3-carboxylic acid according to general method A. ESI-MS: 381 (M + 1).

5- (5-chloro-1 H -Indazol-3-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L4)

Prepared from 5-chloro-1 H -indazole-3-carboxylic acid according to general method A. ESI-MS: 402, 404 (M + 1).

5- (Indol-3-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L26)

Prepared from indole-3-carboxylic acid according to general method A. Indole-3-carboxylic acid was coupled by activation by HOBt (1 equivalent) and DIC (1 equivalent relative to carboxylic acid); Coupling time: 1 hour. ESI-MS: 367 (M + 1).

5- (Benzo [c] isoxazol-3-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L31)

Prepared from benzo [c] isoxazole-3-carboxylic acid according to general method A. ESI-MS: 369 (M + 1).

5- (benzo [c] furan-1-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L32)

Prepared from benzo [c] furan-1-carboxylic acid according to general method A. ESI-MS: 368 (M + 1).

5- (1,5-dimethylpyrazol-3-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L33)

Prepared from 1,5-dimethylpyrazole-3-carboxylic acid according to general method A. ESI-MS: 346 (M + l).

2-methoxy-5- [1-methyl-5- (trifluoromethyl) pyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L34)

Prepared from 1-methyl-5- (trifluoromethyl) pyrazole-3-carboxylic acid according to general method A. ESI-MS: 400 (M + 1).

5- (1-isoquinolinyl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L35)

Prepared from isoquinoline-1-carboxylic acid according to general method A. ESI-MS: 379 (M + 1).

5- (indazol-3-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L36)

Prepared from indazole-3-carboxylic acid according to general method A. Indazole-3-carboxylic acid was coupled by activation by HOBt (1 equivalent) and DIC (1 equivalent relative to carboxylic acid); Coupling time: 1 hour. ESI-MS: 368 (M + 1).

2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido] benzoic acid (3-amino-1-propyl) amide (L42)

Prepared from 5- (2-methylthiazol-4-yl) isoxazole-3-carboxylic acid according to general method A. ESI-MS: 416 (M + 1).

2-methoxy-5- (1-methylpyrazolo [3,4-b] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L44)

Prepared from 1-methylpyrazolo [3,4-b] pyridine-3-carboxylic acid according to general method A. ESI-MS: 383 (M + l).

5- [5- (2-furyl) isoxazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L45)

Prepared from 5- (2-furyl) isoxazole-3-carboxylic acid according to general method A. ESI-MS: 385 (M + 1).

2-methoxy-5- (pyrazolo [1,5-a] pyridin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L46)

Prepared from pyrazolo [1,5-a] pyridine-2-carboxylic acid according to general method A. ESI-MS: 368 (M + 1).

5- (1 H -Benzimidazole-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L47)

Prepared from 1 H -benzimidazole-2-carboxylic acid according to general method A. ESI-MS: 368 (M + 1).

5- (imidazo [1,2-b] pyridazin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L48)

Prepared from imidazo [1,2-b] pyridazine-2-carboxylic acid according to general method A. ESI-MS: 369 (M + 1).

2-methoxy-5- (3-phenylisoxazole-5-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L51)

Prepared from 3-phenylisoxazole-5-carboxylic acid according to general method A. ESI-MS: 395 (M + 1).

2-methoxy-5- (pyrazolo [1,5-a] pyrimidin-2-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L56)

Prepared from pyrazolo [1,5-a] pyrimidine-2-carboxylic acid according to general method A. ESI-MS: 369 (M + 1).

5- (imidazo [1,2-a] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L57)

Prepared from imidazo [1,2-a] pyridine-2-carboxylic acid hydrobromide according to general method A. An additional 1 equivalent of DIPEA was added when the carboxylic acid hydrobromide was coupled using HATU / DIPEA. ESI-MS: 368 (M + 1).

5- (imidazo [1,2-a] pyrimidin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L63)

Prepared from imidazo [1,2-a] pyrimidine-2-carboxylic acid according to general method A. ESI-MS: 369 (M + 1).

General Method B: Synthesis of 5- [3- (aryl or cyclopropyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N- (3-aminopropyl) amide by Suzuki coupling (L12, L13, L14, L22, L23)

For 0.1 mmol of 1,3-diaminopropane-trityl-polystyrene resin, N- Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid are coupled according to general method A It became. The resulting resin loaded with bromopyridine carboxylic amide was thoroughly dried and transferred to a glass vial. Cesium carbonate or potassium carbonate (0.3 mmol) and each boronic acid (1 mmol) were added, followed by addition of absolute DMF (1 mL) and the mixture was flushed extensively with argon. Tetrakis-triphenylphosphinepalladium (0) (10 μmol) was then added, the mixture was again flushed with argon and the vial tightly sealed. The mixture was heated to 100 ° C and shaken overnight. Thereafter, the resin was washed thoroughly (DMF, dichloromethane, each solvent several times) and the small sample was broken down (conditions, see below). If LCMS showed incomplete conversion, the coupling step was repeated or performed at higher temperature. Otherwise, the resin was treated with dichloromethane-TFA-triethylsilane 85: 10: 5 (twofold cleavage), after which the resin was washed with dichloromethane. After evaporation to dryness, the residue was purified by preparative reverse phase HPLC.

2-methoxy-5- {3- [4- (trifluoromethyl) phenyl] pyridin-5-yl} carboxamidobenzoic acid N -(3-aminopropyl) amide (L12)

Prepared from 4- (trifluoromethyl) phenylboronic acid according to general method B. ESI-MS: 473 (M + l).

5- [3- (2-furyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L13)

Prepared from 2-furylboronic acid according to general method B. ESI-MS: 395 (M + 1).

5- [3- (2-benzothienyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L14)

Prepared from 2-benzothienylboronic acid according to general method B. ESI-MS: 461 (M + 1).

5- [3- (3-furyl) pyridin-5-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L22)

Prepared from 3-furylboronic acid according to general method B. ESI-MS: 395 (M + 1).

5- (3-Cyclopropylpyridin-5-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L23)

Prepared from cyclopropylboronic acid according to general method B. ESI-MS: 369 (M + 1).

5- (3-ethynylpyridin-5-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L24)

For 0.1 mmol of 1,3-diaminopropane-trityl-polystyrene resin, N- Fmoc-5-amino-2-methoxybenzoic acid and 5-bromopyridine-3-carboxylic acid are coupled according to general method A It became. The resulting resin loaded with bromopyridine carboxylic amide was thoroughly dried and transferred to a glass vial. Copper (I) chloride (40 μmol), triphenylphosphine (40 μmol), bis (triphenylphosphine) palladium (II) dichloride (10 μmol) and THF (1 mL) were added and the mixture was converted to argon. Flushed. Then trimethylsilylacetylene (1 mmol) and triethylamine (0.5 mL) were added and the mixture was again flushed with argon, then the vial was tightly sealed and shaken at 50 ° C. overnight. The resin was then washed thoroughly with DMF and dichloromethane (each solvent several times). For TMS deprotection, THF (1 mL) and water (50 μL) were added followed by tetrabutylammonium fluoride (1 M solution in THF, 0.5 mL). The mixture was stirred for 2 hours and then the resin was washed with DMF and dichloromethane (each solvent several times). Degradation of the target compound from the resin was effective by treatment with dichloromethane-TFA-triethylsilane 85: 10: 5 (twofold cleavage) followed by washing with dichloromethane. After evaporation, the residue was purified by preparative reverse phase HPLC. ESI-MS: 353 (M + 1).

General Method C: Synthesis of 5- [5- (2-thienyl or 2-furyl) -1-methylpyrazol-3-yl] carboxamidoaryl carboxylic acid N- (3-aminopropyl) amide (L19, L9, L10, L17, L18)

Each ( N- Fmoc protected) aminoarylcarboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent to carboxylic acid) are dissolved in DMF, NMP, DMF / DMSO or NMP / DMSO mixture (1-3 mL), Treated with DIC (1 equivalent to carboxylic acid). After stirring for 2-5 minutes, the mixture was added to 0.1-0.15 mmol of 1,3-diaminopropane linked to trityl-polystyrene resin or 2-chlorotrityl polystyrene resin. The mixture was shaken for several hours or overnight. Next, the resin was washed (DMF or DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times) and treated with 25% piperidine in DMF (1-5 mL) for 15-30 minutes. It was. The resin was then washed thoroughly (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times) and dried in air, in a nitrogen stream, or in high vacuum. 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid or 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.2-0.5 mmol) and HOAt (1 equivalent to carboxylic acid ) Was dissolved in DMF, NMP, DMF / DMSO or NMP / DMSO mixture (1-3 mL) and treated with DIC (1 equivalent to carboxylic acid). After stirring for 2-5 minutes, the mixture was added to the resin and shaken for several hours or overnight. After washing (DMF, dichloromethane or DMF, methanol, dichloromethane, each solvent several times), the target compound is subjected to a suitable decomposition mixture (dichloromethane, TFA, triethylsilane, 85: 10: 5 for trityl resins). , 45:45:10) for 2-chlorotrityl resin) to decompose from the support. Typically, the decomposition step was repeated once, followed by washing the resin with dichloromethane. After evaporation of the solvent, the crude residue was purified by preparative reverse phase HPLC.

2- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidothiazole-4-carboxylic acid N -(3-aminopropyl) amide (L19)

Prepared from N- Fmoc-2-aminothiazole-4-carboxylic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid according to general method C. ESI-MS: 391 (M + 1).

2-Methyl-3- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L9)

Prepared from N- Fmoc-3-amino-2-methylbenzoic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid according to general method C. ESI-MS: 398 (M + 1).

4-Methyl-3- [1-methyl-5- (2-thienyl) pyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L10)

Prepared from N- Fmoc-3-amino-4-methylbenzoic acid and 1-methyl-5- (2-thienyl) pyrazole-3-carboxylic acid according to general method C. ESI-MS: 398 (M + 1).

3- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L17)

Prepared from N- Fmoc-3-aminobenzoic acid and 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid according to general method C. ESI-MS: 368 (M + 1).

4- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamidobenzoic acid N -(3-aminopropyl) amide (L18)

Prepared from N- Fmoc-4-aminobenzoic acid and 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid according to general method C. ESI-MS: 368 (M + 1).

3- { N -[5- (2-furyl) pyrazol-3-yl] carbamoyl} benzoic acid  N'- (3-aminopropyl) amide (L21)

1,3-diaminopropane-trityl resin (0.1 mmol) was expanded in dichloromethane and treated with excess DIPEA and m -benzenedicarboxylic dichloride. After a few minutes, the resin was washed quickly with dichloromethane (three times) and immediately treated with excess 3-amino-5- (2-furyl) pyrazole and DIPEA in NMP. If LCMS (conditions, see below) of the degraded small resin sample showed complete conversion, the resin was washed (DMF, dichloromethane, each solvent several times) and treated with dichloromethane-TFA-triethylsilane ( 85: 10: 5; two-minute method, followed by washing the resin with dichloromethane). The degradation solution was evaporated to dryness and the residue was purified by preparative reverse phase HPLC. ESI-MS: 354 (M + l).

5- (imidazo [2,1-b] thiazol-6-yl) carboxamido-2-methoxybenzoic acid N -(3-aminopropyl) amide (L37)

1,3-Diaminopropane-trityl-resin (0.15 mmol) was pre-expanded in NMP. N- Fmoc-5-amino-2-methoxybenzoic acid (0.2 mmol) and HATU (0.2 mmol) were dissolved in NMP (1.5 mL) and treated with DIPEA (0.4 mmol). After 2 minutes, the solution was added to the resin and the reaction mixture was shaken for 30 minutes. After washing the resin with DMF (three times), 25% piperidine in DMF was added and shaken continuously for 30 minutes. The resin was then washed thoroughly with DMF, methanol and dichloromethane (3 times each solvent) and dried in air.

Imidazo [2,1-b] thiazole-6-carboxylic acid hydrobromide (0.2 mmol) and HATU (0.2 mmol) were dissolved or suspended in NMP (1.5 mL) and treated with DIPEA (0.8 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. After washing the resin with DMF, methanol and dichloromethane (3 times each solvent), the target compound was treated with dichloromethane-TFA-triethylsilane 85: 10: 5 (two-way method) followed by washing with dichloromethane. By decomposition from the resin. After evaporation, the residue was purified by preparative reverse phase HPLC.

ESI-MS: 374 (M + 1).

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-methoxybenzoic acid N -(3-aminopropyl)- N -Methylamide (L28)

2-chlorotrityl chloride resin (150 μmol) was pre-expanded in NMP and treated with NMP (1.5 mL), followed by 3-aminopropanol (1.5 mL). After shaking the resin for 2 hours, it was washed with DMF, methanol and dichloromethane (each solvent several times) and dried in air. Dichloromethane (2 mL) and DIPEA (2 mmol) were then added, followed by dropwise addition of methanesulphonic chloride (1 mmol). The resin was stirred for 2 hours, then washed quickly with dichloromethane (3 times) and expanded in NMP (1 mL). After addition of the methylamine solution (8 M in ethanol, 1 mL), the resin was shaken overnight, then washed with DMF, methanol and dichloromethane (each solvent several times) and dried in air.

N- Fmoc-5-amino-2-methoxybenzoic acid (250 μmol) and HOAt (250 μmol) were dissolved in NMP (about 1.5 mL) and treated with DIC (250 μmol). After 2 minutes, the solution was added to the resin and shaken for 5 hours. After washing with DMF and dichloromethane (each solvent several times), the resin was treated with 25% piperidine in DMF (30 minutes, then washed as described above).

5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (250 μmol) and HOAt (250 μmol) were dissolved in NMP (about 1.5 mL) and treated with DIC (250 μmol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin was then washed as described above, and the target compound was decomposed from the resin by treatment with dichloromethane-TFA-triethylsilane 45:45:10 (two-way method), followed by washing with dichloromethane. After evaporation, the residue was purified by preparative reverse phase HPLC. ESI-MS: 412 (M + l).

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-ethoxybenzoic acid N -(3-aminopropyl) amide (L29)

5-nitrosalicylic acid (0.3 mmol) and HOBt (0.3 mmol) were dissolved in DMSO (about 1.5 mL) and treated with DIC (0.3 mmol). After 2 minutes, the solution was added to 1,3-diaminopropane-trityl resin (0.15 mmol) and shaken for 1 hour, resulting in the resin being DMF, water, diluted sodium carbonate solution, water, methanol and Wash with dichloromethane (each solvent several times). Small resin samples were treated with benzoyl chloride and DIPEA, washed and digested (conditions, see below), and the product was analyzed by HPLC-MS and showed incomplete loading. Thus, the coupling step was repeated for 30 minutes. To hydrolyze the amount of carbamoyl-nitrophenylester formed during that time, the resin was then treated with THF (1 mL), methanol (0.5 mL) and aqueous NaOH (1 M, 0.5 mL) for 5 minutes, yellow The supernatant turned into a successful ester hydrolysis. After washing with DMF, methanol and dichloromethane (3 times each solvent), DMSO (2 mL), cesium carbonate (1 mmol) and ethyl bromide (1 mmol) were added, the resin was shaken for about 2 hours and then the reaction Was repeated overnight (0.2 mL of water was added to dissolve the carbonate salt). Since the conversion was still incomplete, the reaction was repeated with 0.5 mL of ethyl bromide and water (0.2 mL), which led to sufficient reaction progress after 3 days, after which the resin was converted to DMF, water, methanol and dichloromethane. Washed (each solvent several times).

The nitro group was reduced by addition of a solution of tin (II) chloride (1 mmol) in NMP (1 mL), pyridine (0.5 mL) and NMP (1 mL). After the mixture was shaken overnight, the resin was washed with DMF, water and methanol and insoluble byproducts were carefully removed by flotation in methanol. After washing with dichloromethane, the resin was dried. 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.25 mmol) and HOAt (0.25 mmol) were dissolved in NMP (about 1.5 mL) and treated with DIC (0.25 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin is then washed with DMF and dichloromethane (each solvent several times) and the target compound is treated with dichloromethane-TFA-triethylsilane 85: 10: 5 (two-way method) followed by washing with dichloromethane. By decomposition from the resin. After evaporation, the residue was purified by preparative reverse phase HPLC. ESI-MS: 412 (M + l).

5- [5- (2-furyl) -1-methylpyrazol-3-yl] carboxamido-2-hydroxybenzoic acid N -(3-aminopropyl) amide (L30)

5-nitrosalicylic acid (0.3 mmol) and HOBt (0.3 mmol) were dissolved in DMSO (about 1.5 mL) and treated with DIC (0.3 mmol). After 2 minutes, the solution was added to 1,3-diaminopropane-trityl resin (0.15 mmol) and shaken for 1 hour, resulting in the resin being DMF, water, diluted sodium carbonate solution, water, methanol and dichloro Washed with methane (each solvent several times). Small resin samples were treated with benzoyl chloride and DIPEA, washed and digested (conditions, see below), and the product was analyzed by HPLC-MS and showed incomplete loading. Thus, the coupling step was repeated for 30 minutes. To hydrolyze the amount of carbamoyl-nitrophenylester formed during that time, the resin was then treated with THF (1 mL), methanol (0.5 mL) and aqueous NaOH (1 M, 0.5 mL) for 5 minutes, yellow The supernatant turned into a successful ester hydrolysis. After washing with DMF, methanol and dichloromethane (3 solvents each), DMSO (2 mL), cesium carbonate (1 mmol) and allyl bromide (1 mmol) were added, the resin was shaken for 1 hour, and then the reaction was allowed to proceed. Repeat overnight (0.2 mL of water was added). Since the conversion was still incomplete, the reaction was repeated once more for three days, after which the resin was washed with DMF, water, methanol and dichloromethane (each solvent several times).

The nitro group was reduced by addition of a solution of tin (II) chloride (1 mmol) in NMP (1 mL), pyridine (0.5 mL) and NMP (1 mL). After shaking the mixture overnight, the resin was washed with DMF, water and methanol and insoluble byproducts were carefully removed by suspension in methanol. After washing with dichloromethane, the resin was dried. 5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (0.25 mmol) and HOAt (0.25 mmol) were dissolved in NMP (about 1.5 mL) and treated with DIC (0.25 mmol). After 2 minutes, the solution was added to the resin and shaken overnight. The resin was then washed with DMF and dichloromethane (each solvent several times).

Allyl ether was digested by treating the resin with dichloromethane (2 mL), piperidine (0.4 mL) and tetrakis-triphenylphosphinepalladium (0) (about 10 mg) for about 2 hours. Thereafter, the resin was thoroughly washed with DMF, water, methanol and dichloromethane (each solvent several times), and the target compound was treated with dichloromethane-TFA-triethylsilane 85: 10: 5 (two-way method), It was then decomposed from the resin by washing with dichloromethane. After evaporation, the residue was purified by preparative reverse phase HPLC. ESI-MS: 384 (M + 1).

5- (1-ethylindazol-3-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L38)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 3 hours at room temperature. After washing the resin with DMF, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for about 30 minutes, followed by thorough washing with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and the solution was added to the resin after 2 minutes. The mixture was shaken for 1 hour, after which the resin was washed with DMF and dichloromethane. Cesium carbonate (1 mmol) and dry DMF (1.5 mL) were added to the resin followed by ethyl bromide (1.5 mmol). The mixture was shaken overnight and then washed with DMF, water, methanol and dichloromethane. The cesium carbonate / ethyl bromide step was repeated once over three days, after which the resin was washed thoroughly. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 396 (M + 1).

5- [ N -(1-methylindazol-3-yl) carbamoyl] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L39)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP. 5-Formylsalicylic acid (0.5 mmol) and HOAt (0.5 mmol) were dissolved in NMP (1.5 mL) and treated with DIC (0.5 mmol). After 2 minutes, the solution was added to the resin and the mixture was stirred for 2 hours, as a result of which the resin was washed several times with DMF and DCM. The resin was then treated with THF (1 mL), methanol (1 mL) and aqueous NaOH (2 M, 0.5 mL) for 1 hour at room temperature, after which the resin was treated with methanol / water, DMF, methanol / water, DMF and Washed with DCM. After addition of acetonitrile (1 mL), tert -butanol (1 mL) and 2-methyl-2-butene (200 μL), the resin is cooled to 0 ° C. and monosodium phosphate (0.2 mL) in water (0.2 mL) mmol) and sodium chlorite (0.25 mmol). After 30 minutes, acetonitrile (1 mL) and tert -butanol (1 mL) were added and monosodium phosphate (0.8 mmol) and sodium chlorite (1 mmol) in water (0.4 mL) were added dropwise, the mixture above Warmed to room temperature. The resin was washed with methanol / water, DMF, methanol / water, DMF and DCM. After drying thoroughly, triphenylphosphine (1 mmol), THF (2 mL) and methanol (0.2 mL) were added followed by diethyl azodicarboxylate (1 mmol). The resin was stirred at rt for 1 h and then washed several times with DMF and dichloromethane. THF (1 mL), methanol (1 mL) and aqueous NaOH (2 M, 0.5 mL) are added and the mixture is shaken for 4 days at room temperature, after which the resin is added to methanol / water, DMF, methanol / water, DMF And washed with DCM. A solution of HATU (0.25 mmol) and DIPEA (0.5 mmol) in NMP (1 mL) was added and the mixture was shaken for 15 minutes at room temperature. Then 3-amino-1-methylindazole (0.25 mmol) in NMP (0.5 mL) was added and the reaction mixture was shaken overnight, then washed with DMF and dichloromethane. The target compound was decomposed from the support by two-way treatment with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 382 (M + 1).

( RS ) -5- [1- (2,3-dihydroxy-1-propyl) indazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L40)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 3 hours at room temperature. After washing the resin with DMF, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for about 30 minutes, followed by thorough washing with DMF and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and the solution was added to the resin after 2 minutes. The mixture was shaken for 1 hour, after which the resin was washed with DMF and dichloromethane. Cesium carbonate (1 mmol) and dry DMF (1.5 mL) were added to the resin followed by allyl bromide (1 mmol). The mixture was shaken overnight and then washed with DMF, water, methanol and dichloromethane. The cesium carbonate / allyl bromide step was repeated once overnight, after which the resin was washed thoroughly, dried and transferred to a glass vial. N -methylmorpholine oxide (0.5 mmol) in water (0.5 mL) and acetone (2 mL) was added to the resin. Osmium- (VIII) -oxide (2.5% solution in 50 μL tert -butanol) was added and the vial was tightly sealed and shaken at room temperature overnight. An additional portion of osmium- (VIII) -oxide (2.5% solution in 50 μL tert -butanol) was added and shaken for an additional 2 hours, followed by reaction with aqueous sodium dithionite (174 mg in 1 mL of water). Quench and shake for 30 minutes. The resin was then washed several times with water, DMF, water, methanol, DMF and dichloromethane. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 442 (M + l).

5- (3 H Imidazo [4,5-b] pyridin-2-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L41)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours at room temperature. After washing the resin with DMF and dichloromethane, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for about 30 minutes, followed by thorough washing with DMF and dichloromethane. The resin was expanded in dichloromethane (1 mL), DIPEA (1 mmol) was added followed by oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane (1 mL). After shaking for 5 minutes, the resin was washed with DMF, methanol and dichloromethane (3 times each). 2,3-diaminopyridine (0.5 mmol) in NMP (2 mL) was added and the mixture was shaken at about 100 ° C. for 4 days. After washing the resin (DMF, methanol, dichloromethane), the reaction was repeated at about 120 ° C. overnight. After washing as described above, THF (1 mL), methanol (0.5 mL) and aqueous NaOH (2 M, 0.5 mL) were added to the resin and the mixture was stirred for 15 minutes, resulting in the resin being methanol ( 5 times) and dichloromethane (3 times). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 369 (M + 1).

2-methoxy-5-([1,2,4] triazolo [4,3-a] pyridin-3-ylcarboxamido) benzoic acid (3-amino-1-propyl) amide (L43)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded in NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol), and after 2 minutes the mixture was added to a stirred resin for 3 hours. Added. After washing the resin with DMF, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for 30 minutes. The resin was then washed thoroughly with DMF and dichloromethane and expanded in dichloromethane. Dichloromethane (1.5 mL), DIPEA (1 mmol) and oxalic acid monoethyl ester monochloride (0.5 mmol) were added and the mixture was stirred for 10 minutes before the resin was washed several times with DMF and dichloromethane. Ethylene glycol (1 mL), 2-pyridylhydrazine (1 mmol) and DIPEA (1 mmol) were added and the mixture was heated to 100 ° C. for 4 hours, then the resin was washed several times with DMF and dichloromethane. . Triphenylphosphine (0.5 mmol), N -methylmorpholine (0.5 mmol) and dichloromethane (2 mL) were added to the resin, followed by trichloroacetonitrile (0.4 mmol) was added dropwise. After shaking the mixture for 48 hours, a solution of triphenylphosphine (0.5 mmol) and N -methylmorpholine (1 mmol) in dichloromethane (2 mL) was added together with tetrachlorocarbon (0.5 mL). The mixture was kept at 40 ° C. for 2 hours, then THF (2 mL) and aqueous sodium carbonate (2 M, 0.5 mL) were added and the mixture was stirred overnight. Finally, the resin was washed thoroughly with DMF, water, methanol and dichloromethane. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 369 (M + 1).

( S ) -2,6-bis [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] hexanoic acid (3-amino-1- Propyl) amide (L49)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was expanded in NMP. Thereafter, ( S ) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5 mL) and treated with DIPEA (400 μmol) for 2 minutes, resulting in a solution of resin Was added and the mixture was stirred at room temperature. After 1 hour, the resin was washed with DMF, piperidine (25% in DMF, 2 mL) was added and the resin stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-5-amino-2-methoxybenzoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution to the resin. Add and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Imidazo [2,1-b] thiazole-6-carboxylic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes. The solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 801 (M + l), 401 ((M + 2) / 2).

( S ) -2,6-bis {8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-di Oxaoctanoyl amido} hexanoic acid (3-amino-1-propyl) amide (L50)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was expanded in NMP. Thereafter, ( S ) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5 mL) and treated with DIPEA (400 μmol) for 2 minutes, resulting in a solution of resin Was added and the mixture was stirred at room temperature. After 1 hour, the resin was washed with DMF, piperidine (25% in DMF, 2 mL) was added and the resin stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-8-amino-3,6-dioxaoctanoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution. Add to the resin and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Fmoc-5-amino-2-methoxybenzoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution to the resin. Add and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Imidazo [2,1-b] thiazole-6-carboxylic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes. The solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 547 ((M + 2) / 2).

5- [4- (2-furyl) pyridin-2-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L52)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was preexpanded in NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) and after 2 minutes the solution was added to the resin. After shaking for 105 minutes, the resin was washed with DMF, then treated with piperidine (25% in DMF, 2 mL) for 1 hour, followed by thorough washing with DMF, methanol and dichloromethane. 4-Bromopyridine-2-carboxylic acid (0.3 mmol) and HOAt (0.3 mmol) in NMP (3 mL) were treated with DIC (0.3 mmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 1 hour, as a result the resin was washed as described above, thoroughly dried with a dry nitrogen stream and transferred to a glass vial. Cesium carbonate (0.3 mmol) and 2-furylboronic acid (0.5 mmol) were added with DMF (2 mL). The mixture was flushed thoroughly with argon. After addition of tetrakis-triphenylphosphinylpalladium-0 (10 μmol), the mixture was flushed once more with argon, then the vial was tightly sealed and the mixture was stirred at 100 ° C. overnight. The resin was then washed with DMF, water, methanol and dichloromethane (three times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 395 (M + 1).

( S ) -2,6-bis {5- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3-oxopentanoyl Amido} hexanoic acid (3-amino-1-propyl) amide (L53)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was expanded in NMP. Thereafter, ( S ) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5 mL) and treated with DIPEA (400 μmol) for 2 minutes, resulting in a solution of resin Was added and the mixture was stirred at room temperature. After 1 hour, the resin was washed with DMF, piperidine (25% in DMF, 2 mL) was added and the resin stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-5-amino-3-oxapentanoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution to the resin. Add and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Fmoc-5-amino-2-methoxybenzoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution to the resin. Add and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Imidazo [2,1-b] thiazole-6-carboxylic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes. The solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 503 ((M + 2) / 2).

2-methoxy-5- [3- (4-methoxyphenyl) -1,2,4-oxadiazole-5-ylcarboxamido] benzoic acid (3-amino-1-propyl) amide (L54)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (450 μmol, about 0.5 g) was pre-expanded in dichloromethane and then pre-expanded in NMP. Fmoc-5-amino-2-methoxybenzoic acid (0.6 mmol) and HATU (0.6 mmol) in NMP (4.5 mL) were treated with DIPEA (1.2 mmol) for 2 minutes. Then the solution was added to the resin and the mixture was stirred for 40 minutes and then washed three times with DMF. After addition of piperidine (25% in DMF, about 6 mL), the resin was stirred for 30 minutes and then washed with DMF, methanol and dichloromethane (3 times each). After drying the resin in high vacuum, dichloromethane (3 mL) and DIPEA (3 mmol) were added and oxalic acid monoethyl ester monochloride (1.5 mmol) in dichloromethane was carefully added. After shaking the mixture for 2 minutes, the resin was washed with dichloromethane, methanol and dichloromethane (3 times each). After drying the resin, 150 mg was removed. The remaining amount was treated with THF (4 mL), methanol (2 mL) and aqueous NaOH (2 M, 2 mL) at room temperature for 2 hours. The resin was washed (DMF, acetic acid [2.5% in DMF], methanol and dichloromethane; three times each). In the resin, 150 mg was reacted with HOAt (0.5 mmol) and DIC (0.5 mmol) in NMP (1 mL) for 2 minutes. Then 4-methoxy- N -hydroxybenzamidine (0.5 mmol) in NMP (1 mL) was added. After stirring the resin for 1 hour, DIC (0.5 mmol) was added and the mixture was shaken overnight. After the resin was washed with DMF, methanol and dichloromethane (three times each), the coupling step was repeated. After 3 hours from the second addition of DIC, the resin was washed as described above, dried and transferred to a glass vial. NMP (2 mL) and DIC (0.5 mmol) were added and the mixture was heated to 120 ° C. for 30 minutes. The resin was then washed as described above. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 426 (M + 1).

5- [3- (2-furyl) -1,2,4-oxadiazole-5-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L55)

Synthesis of N-hydroxy-2-furylcarboxamidine: To the stirred solution of 2 -cyanofuran (5 mmol) in ethanol (5 mL) was added aqueous hydroxylamine (50%, 5 mmol), As a result, a slight exotherm of the reaction mixture was observed. After about 20 minutes, additional aqueous hydroxylamine (0.5 mmol) was added. After stirring for 1 hour, the solvent was evaporated with a stream of nitrogen and the residue was purified by flash column chromatography (NH ₂ -modified stationary phase, 0-5% methanol in dichloromethane, 1% triethylamine), A slightly turbid oil was provided which solidified when standing still.

Synthesis of Target Compound: Dry 2-chlorotrityl chloride resin (150 μmol) was treated with 1,3-diaminopropane (2 mL) followed by dichloromethane (2 mL). After 5 minutes, the resin was washed with DMF, methanol and dichloromethane (3 times each) and expanded in NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol), and after 2 minutes the solution was shaken at room temperature for 30 minutes. Was added. The resin was washed with DMF (3 times) and treated with piperidine (25% in DMF, about 4 mL) for 30 minutes, as a result of washing the resin with DMF, methanol and dichloromethane (3 times each), Expanded in dichloromethane. After addition of dichloromethane (2 mL) and DIPEA (1 mmol), a solution of oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane (1 mL) was carefully added and the mixture was stirred for 5 minutes, As a result, the resin was washed with DMF, methanol and dichloromethane (3 times each), dried and transferred to a glass vial. Potassium carbonate (0.5 mmol), toluene (2 mL) and N -hydroxy-2-furylcarboxamidine (0.5 mmol; see above) were added, the vial was tightly sealed and stirred at 100 ° C. overnight. And stirred at 110 ° C. for 2 hours, as a result of washing the resin with DMF, methanol and dichloromethane (3 times each). The target compound was subjected to dichotomy with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane and removal of the solvent with a stream of nitrogen from the support. Digested. The residue was purified by preparative reverse phase HPLC. ESI-MS: 386 (M + 1).

5- [5- (2-furyl) -1,3,4-oxadiazol-2-yl] carboxamido-2-methoxybenzoic acid (3-amino-1-propyl) amide (L58)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP, followed by removal of excess solvent. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the expanded resin and the mixture was shaken for 2 hours at room temperature. After washing the resin with DMF, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for about 30 minutes, followed by thorough washing with DMF and dichloromethane. The resin was then expanded in dichloromethane and treated with oxalic acid monoethyl ester monochloride (0.5 mmol) and DIPEA (1 mmol) in dichloromethane (1.5 mL) for 10 minutes, followed by repeated washing with DMF and dichloromethane. . The resin was expanded in THF (1.5 mL), then hydrazine hydrate (0.5 mL) was added and the resin was stirred at room temperature for 3 hours, then washed with DMF and dichloromethane. Furan-2-carboxylic acid (0.5 mmol) and HOAt (0.5 mmol) were dissolved in NMP (1.5 mL) and treated with DIC (0.5 mmol). After 2 minutes, the solution was added to the resin and the mixture was stirred for 2 hours at room temperature, then washed with DMF and dichloromethane. Then a solution of triphenylphosphine (0.5 mmol) and N -methylmorpholine (1 mmol) in dichloromethane (2 mL) was added followed by tetrachlorocarbon (0.5 mL). The mixture was heated to 40 ° C. for 2 hours in a tightly sealed glass vial. After addition of THF (2 mL) and aqueous Na ₂ CO ₃ (2 M, 0.5 mL), the mixture was stirred at rt overnight, then washed with DMF, water, methanol and dichloromethane. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 386 (M + 1).

5- [1- (2-hydroxyethyl) indazol-3-ylcarboxamido] -2-methoxybenzoic acid (3-amino-1-propyl) amide (L59)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded with NMP. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 1 hour at room temperature. After washing the resin with DMF, piperidine (25% in DMF, about 2 mL) was added and the resin was shaken for 1 hour, followed by thorough washing with DMF, methanol and dichloromethane. Indazole-3-carboxylic acid (0.5 mmol) and HOBt (0.5 mmol) were dissolved in NMP (1.5 mL). DIC (0.5 mmol) was added and after 2 minutes the solution was added to the resin. The mixture was shaken for 40 minutes, after which the resin was washed with DMF, methanol and dichloromethane (three times each). The resin was transferred to a glass vial and a solution of bromoacetic acid methyl ester (0.5 mmol) and DIPEA (1 mmol) in NMP (2 mL) was added. The vial was tightly sealed and shaken at 80 ° C. for 75 minutes. The resin was then washed (DMF, methanol, dichloromethane, three times each), expanded in THF (2 mL) and methanol (0.5 mL), sodium borohydride (very excess, added in portions). Treated with. After the last addition, the mixture was shaken for 30 minutes at room temperature, then the resin was washed with methanol and dichloromethane (three times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 412 (M + l).

8- [2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamido] -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L60 )

Trityl polystyrene preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded in NMP. Fmoc-8-amino-3,6-dioxaoctanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) and after 2 minutes the solution was allowed to stand at room temperature for 1 hour. The shaker was added to the resin and the result was washed with DMF (3 times). Piperidine (25% in DMF, about 2 mL) was added and the mixture was shaken for about 30 minutes. The resin was washed with DMF, methanol and dichloromethane (three times each). Next, a coupling / deprotection protocol was applied to Fmoc-5-amino-2-methoxybenzoic acid (the same amount as described above). Finally, 1-methylindazole-3-carboxylic acid was coupled using the HATU coupling protocol described above. The resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 527 (M + l).

8- {8- [2-methoxy-5- (1-methylindazol-3-ylcarboxamido) phenylcarboxamido] -3,6-dioxaoctanoylamido} -3,6- Dioxaoctanoic acid (3-amino-1-propyl) amide (L61)

Trityl polystyrene preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded in NMP. Fmoc-8-amino-3,6-dioxaoctanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) and after 2 minutes the solution was allowed to stand at room temperature for 1 hour. The shaker was added to the resin and the result was washed with DMF (3 times). Piperidine (25% in DMF, about 2 mL) was added and the mixture was shaken for about 30 minutes. The resin was washed with DMF, methanol and dichloromethane (three times each). The coupling / deprotection protocol was then repeated with Fmoc-8-amino-3,6-dioxaoctanoic acid, which was then applied to Fmoc-5-amino-2-methoxybenzoic acid (the same amount as described above). It was. Finally, 1-methylindazole-3-carboxylic acid was coupled using the HATU coupling protocol described above. The resin was washed with DMF, methanol and dichloromethane (three times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 672 (M + l).

( S ) -2,6-bis {8- [5- (imidazo [1,2-b] pyridazine-2-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-di Oxaoctanoyl amido} hexanoic acid (3-amino-1-propyl) amide (L62)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was expanded in NMP. ( S ) -bis-Fmoc-lysine (200 μmol) and HATU (200 μmol) were dissolved in NMP (about 1.5 mL), treated with DIPEA (400 μmol) for 2 minutes, and the solution was added to the resin and The mixture was stirred at room temperature. After 1 hour, the resin was washed with DMF, piperidine (25% in DMF, 2 mL) was added and the resin stirred for 30 minutes. The resin was then washed (DMF, methanol and dichloromethane). Fmoc-8-amino-3,6-dioxaoctanoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution. Add to the resin and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Fmoc-5-amino-2-methoxybenzoic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes, resulting in solution to the resin. Add and shake the mixture for 1 hour. Deprotection was achieved by washing the resin with DMF followed by treatment with 25% piperidine in DMF and then washing as described above. Imidazo [1,2-b] pyridazine-2-carboxylic acid (400 μmol) and HATU (400 μmol) were dissolved in NMP (about 2 mL) and treated with DIPEA (800 μmol) for 2 minutes. The solution was added to the resin. After shaking the mixture for 1 hour, the resin was washed with DMF, methanol and dichloromethane (3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 542 ((M + 2) / 2).

( S ) -1-aminopropane-1,3-dicarboxylic acid bis {3- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido ] Propyl} amide (L64)

Synthesis of 5-amino-2-methoxybenzoic acid methyl ester: palladium (0) and 2-methoxy-5 on charcoal (5%, 25 mg [containing about 50% w / w water]) in methanol (5 mL) Nitrobenzoic acid methyl ester (1 mmol) was added dropwise over 10 minutes with triethylsilane (1 mL) followed by methanol (2 mL). After stirring for an additional 5 minutes, the mixture was filtered through celite and then the celite layer was thoroughly washed. The filtrate was concentrated, dried in high vacuum and the residue was used without further purification.

Solution-phase synthesis of aminopropyl-linked ligands (same as L37): imidazo [2,1-b] thiazole-6-carboxylic acid (1 mmol) and TBTU (1 mmol) in DMF (2 mL) Treated with DIPEA (2 mmol) for min. The mixture was then added to the previously obtained complete amount of 5-amino-2-methoxybenzoic acid methyl ester and the mixture was stirred for 30 minutes. After addition of saturated aqueous sodium carbonate (1 mL) and water (3 mL), the mixture was extracted four times with ethyl acetate. The combined organic layers were washed twice with aqueous citric acid (5%), once with saturated aqueous sodium carbonate, and twice with water. The remaining organic layer was concentrated and dried overnight with a dry nitrogen stream. 1,3-diaminopropane (2 mL) was added to the residue and the mixture was heated to 80 ° C for 4 h. The solution was then concentrated to dryness and the residue was redissolved in methanol, concentrated, dried in high vacuum, and provided with an oil which solidified upon refrigeration. Yield: 453 μmol (45%).

Synthesis of Target Compound: N- Boc-glutamic acid (227 μmol) and TBTU (453 μmol) were dissolved in DMF (2 mL), treated with DIPEA (906 μmol) for 3 minutes, and the solution previously obtained It was added to the residue. After 90 minutes another portion of TBTU-activated N- Boc-glutamic acid (0.2 mmol) in DMF (1 mL; activated for 2 minutes as described above) was added. After 45 minutes, the solvent was evaporated overnight into a nitrogen stream. The residue was redissolved in methanol, evaporated to dryness and treated with trifluoroacetic acid (25% in dichloromethane) followed by evaporation to dryness and purification of the residue by preparative reverse phase HPLC. ESI-MS: 858 (M + l), 430 ((M + 2) / 2).

General Method D: Synthesis of 5-amino-2-methoxybenzoic acid linked to two Ado units and one 1,3-diaminopropane unit (basic resin)

Trityl polystyrene resin preloaded with 1,3-diaminopropane was pre-expanded in DCM and NMP. Fmoc-8-amino-3,6-dioxaoctanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes, then the solution was added to the resin and The mixture was stirred for 30 minutes to several hours. After washing the resin with DMF or DMF, methanol and dichloromethane, piperidine (25% in DMF, about 2 mL) was added and the resin stirred for at least 20 minutes. The resin was then washed thoroughly with DMF, methanol and dichloromethane (3 times each) and the coupling / deprotection process was repeated once. Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) was coupled to the resin using the HATU-coupling protocol described above and deprotected with piperidine as described above. The resin was dried in air prior to further synthesis steps.

8- {8- {5- [5- (2-furyl) -1-methylpyrazol-3-ylcarboxamido] -2-methoxyphenylcarboxamido} -3,6-dioxaoctanoyl Amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L65)

5- (2-furyl) -1-methylpyrazole-3-carboxylic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes. The solution was then added to the base resin obtained by general method D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, 3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 688 (M + 1).

8- {8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoylamido } -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L66)

Imidazo [2,1-b] thiazole-6-carboxylic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes. The solution was then added to the base resin obtained by general method D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, 3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 664 (M + l).

8- {8- {5- [5- (2-furyl) -1,3,4-oxadiazole-2-ylcarboxamido] -2-methoxyphenylcarboxamido} -3,6- Dioxaoctanoyl amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L67)

The dry base resin obtained by general method D was treated with dichloromethane (2 mL) and DIPEA (1 mmol), followed by dropwise addition of oxalic acid monoethyl ester monochloride (0.5 mmol) in dichloromethane. The mixture was stirred at room temperature for 10 minutes, after which the resin was washed with DMF, methanol and dichloromethane (three times each). The resin was then expanded in NMP (2 mL) and treated with hydrazine hydrate (1 mmol) for 50 minutes at room temperature and then washed as described above. 2-furoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes, then the solution was added to the resin. After about 1 hour, the resin was washed as described above. Next, 4-toluenesulfonyl chloride (0.5 mmol) and DIPEA (1 mmol) in dichloromethane (about 2 mL) were added and the mixture was stirred until cyclization was complete. The resin was washed as described above. The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 676 (M + l).

8- {8- {2-methoxy-5- [5- (2-methylthiazol-4-yl) isoxazol-3-ylcarboxamido] phenylcarboxamido} -3,6-dioxa Octanoyl amido} -3,6-dioxaoctanoic acid (3-amino-1-propyl) amide (L68)

5- (2-methylthiazol-4-yl) isoxazole-3-carboxylic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes. The solution was then added to the base resin obtained by general method D. After stirring the mixture for about 2 hours, the resin was washed (DMF, methanol, dichloromethane, 3 times each). The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 706 (M + l).

N -(8-amino-3,6-dioxaoctyl)- N'- {8- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctyl} urea (L69)

To 2-chlorotrityl chloride polystyrene resin (150 μmol) was added 1,8-diamino-3,6-dioxaoctane (2 mL). After shaking the mixture for 5 minutes, dichloromethane (1 mL) was added to ensure sufficient expansion. After 25 minutes, the resin was washed (DMF, methanol, dichloromethane, three times each). The resin was expanded in dichloromethane, then dichloromethane (2 mL) and DIPEA (1 mmol) were added followed by chloroformic acid (4-nitrophenyl) ester (0.5 mmol) in dichloromethane (1 mL). The mixture was stirred for 5 minutes, then the resin was filtered and immediately treated with 1,8-diamino-3,6-dioxaoctane (2 mL) for 20 minutes. Thereafter, the resin was washed as described above.

Fmoc-5-amino-2-methoxybenzoic acid (200 μmol) and HATU (200 μmol) in NMP / DCM (1.5 + 1.5 mL) were treated with DIPEA (400 μmol) for 2 minutes, and then the solution was added to the resin. Added. After stirring the mixture for 5 hours, the resin was washed as described above, treated with piperidine (25% in DMF, about 2 mL) for 30 minutes, then washed as described above. Finally, imidazo [2,1-b] thiazole-6-carboxylic acid ((200 μmol) was coupled using the HATU coupling protocol as described above (neat NMP, about 1.5 mL; reaction time: Overnight), then the resin was washed as described above The target compound was subjected to dichotomy with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, and then the resin was thoroughly washed with dichloromethane. The crude product was purified by reverse phase HPLC after evaporation of the solvent ESI-MS: 622 (M + 1), 312 ((M + 2) / 2).

General Method E: Isophthalic Acid N -(3-amino-1-propyl)- N ' Synthesis of -aryl amides

Dry trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was treated with isophthaloyl chloride (1 mmol) and DIPEA (2 mmol) in dichloromethane (1.5 mL) for 3 minutes. The resin was washed quickly with dichloromethane or NMP (twice or three times). Then, each aromatic amine (0.3 mmol) and DIPEA (0.3 mmol) in NMP (1.5 mL) were added and the mixture was stirred for 1 h and then washed with DMF, methanol and dichloromethane (3 times each) . The target compound was decomposed from the support by dichotomying with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane and then washing the resin thoroughly with dichloromethane. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. Because of the somewhat inferior solubility of the compound, in some cases it was necessary to add methanol to the decomposition or washing solution.

Isophthalic acid N -(3-aminopropyl)- N'- [5- (2-furyl) -1,3,4-thiadiazol-2-yl] amide (L70)

Prepared from 2-amino-5- (2-furyl) -1,3,4-thiadiazole according to general method E. ESI-MS: 372 (M + l).

Isophthalic acid N -(3-aminopropyl)- N'- (1-methylindazol-3-yl) amide (L71)

Prepared from 3-amino-1-methylindazole according to general method E. ESI-MS: 352 (M + l).

Isophthalic acid N -(3-aminopropyl)- N'- (Benzothiazol-2-yl) amide (L72)

Prepared from 2-aminobenzothiazole according to general method E. ESI-MS: 355 (M + 1).

Isophthalic acid N -(3-aminopropyl)- N'- (4-phenylthiazol-2-yl) amide (L73)

Prepared from 2-amino-4-phenylthiazole according to general method E. ESI-MS: 381 (M + 1).

Isophthalic acid N -(3-aminopropyl)- N'- (5-phenylthiazol-2-yl) amide (L74)

Prepared from 2-amino-5-phenylthiazole according to general method E. ESI-MS: 381 (M + 1).

5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxybenzoic acid [3- (6-aminohexanoylamido) -1-propyl] amide (L75)

5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxybenzoic acid (3-amino-1-propyl) amide (L37) was imidazo [ Prepared from 2,1-b] thiazole-6-carboxylic acid and 150 μmol of 1,3-diaminopropane-preloaded trityl polystyrene resin. The crude product obtained by acid decomposition of the support binding ligand was dried over a stream of nitrogen. Saturated aqueous sodium carbonate (3 mL) was added and the mixture was extracted with ethyl acetate (3 times) and dichloromethane (4 times). Combined organic layers were evaporated. Boc-6-aminohexanoic acid (100 μmol) and HATU (100 μmol) in DMF (1 mL) were treated with DIPEA (200 μmol) and after 2 minutes the solution was added to the previously obtained residue 20 minutes After stirring the mixture for 2 h, water (2 mL) and saturated aqueous sodium carbonate (1 mL) were added and the mixture was extracted twice with ethyl acetate. The combined organic layers were washed with aqueous citric acid (5%, twice), saturated sodium carbonate and water and concentrated to dryness. The residue was treated with dichloromethane (1 mL) and trifluoroacetic acid (1 mL) for 40 minutes. After evaporation of the solvent, the crude product was purified by reverse phase preparative HPLC. ESI-MS: 487 (M + 1).

6- [5- (imidazo [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] hexanoic acid (8-amino-3,6-dioxa- 1-octyl) amide (L76)

2-chlorotrityl chloride polystyrene resin (150 μmol) was treated with 1,8-diamino-3,6-dioxaoctane (500 μL). After shaking the mixture for 3 minutes, dichloromethane (1 mL) was added and shaken continuously for 15 minutes, followed by washing with DMF, methanol and dichloromethane (3 times each). The resin was then expanded in NMP. Fmoc-6-aminohexanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours. The resin was then washed as described above and then treated with piperidine (25% in DMF, about 2 mL) for 1 hour and washed as described above. Following this coupling / deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 minutes) followed by imidazo [2,1 -b] coupling of thiazole-6-carboxylic acid was carried out (coupling time: 3 h). After the final wash as described above, the target compound is subjected to dichotomy with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane, Decomposition from the support. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 561 (M + l).

8- [5- (imidazo- [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoic acid (3-amino- 1-propyl) amide (L77)

Trityl polystyrene resin preloaded with 1,3-diaminopropane (150 μmol) was pre-expanded in NMP. Fmoc-8-amino-3,6-dioxaoctanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was stirred for 2 hours. The resin was then washed as described above, then washed with piperidine (25% in DMF, about 2 mL) for 1 hour and washed as described above. Following this coupling / deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 minutes) followed by imidazo [2,1 -b] coupling of thiazole-6-carboxylic acid was carried out (coupling time: 3 h). After the final wash as described above, the target compound is subjected to dichotomy with trifluoroacetic acid (10%) and triethylsilane (5%) in dichloromethane, followed by thorough washing of the resin with dichloromethane, Decomposition from the support. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 519 (M + l).

8- [5- (imidazo- [2,1-b] thiazole-6-ylcarboxamido) -2-methoxyphenylcarboxamido] -3,6-dioxaoctanoic acid (6-amino- 1-hexyl) amide (L78)

2-chlorotrityl chloride polystyrene resin (150 μmol) was treated with 1,6-diaminohexane (500 μL). After shaking the mixture for 3 minutes, dichloromethane (1 mL) was added and shaken continuously for 15 minutes, followed by washing with DMF, methanol and dichloromethane (3 times each). The resin was then expanded in NMP. Fmoc-8-amino-3,6-dioxaoctanoic acid (200 μmol) and HATU (200 μmol) in NMP (1.5 mL) were treated with DIPEA (400 μmol). After 2 minutes, the solution was added to the resin and the mixture was shaken for 2 hours. The resin was then washed as described above and then treated with piperidine (25% in DMF, about 2 mL) for 1 hour and washed as described above. Following this coupling / deprotection cycle, Fmoc-5-amino-2-methoxybenzoic acid was coupled to the resin (coupling time: overnight; deprotection time: 30 minutes) followed by imidazo [2,1 -b] coupling of thiazole-6-carboxylic acid was carried out (coupling time: 3 h). After the final wash as described above, the target compound is subjected to dichotomy with trifluoroacetic acid (45%) and triethylsilane (10%) in dichloromethane, followed by thorough washing of the resin with dichloromethane, Decomposition from the support. After evaporation of the solvent, the crude product was purified by reverse phase HPLC. ESI-MS: 561 (M + l).

Example 3: Immobilization of Ligands

Experiment

The dried ligand was redissolved in DMSO at an approximate concentration of 100 μmM based on gravimetric analysis. The exact concentration of the stock solution was measured by the o-phthaldialdehyde assay (2). Dye-ligand molecules were quantified by measuring absorbance at 340 nm. Measurements were calibrated with 1- (3-aminopropyl) imidazole. In the coupling reaction, one volume of ligand dissolved in 90% DMSO and 10% N-methyl-2-pyrrolidone containing 1M N, N-diisopropylethylamine at an approximate concentration of 15-20 MM isopropanol Was added to 1 volume of standing NHS-activated Sepharose 4 FF (GE Healthcare) equilibrated with. The reaction was vigorously shaken while running at 25 ° C. for 2 hours. The supernatant of the reaction was removed and the resin washed twice with the appropriate solvent. The active groups remaining in the resin were blocked by 1 [mu] M ethanolamine at 25 [deg.] C for 1 hour. The final resin was washed and stored in 20% ethanol at 4 ° C. until used in subsequent experiments. Reaction yield was determined based on mass balance after analyzing the ligand concentration in all fractions.

Summary and Results

The ligand of Example 2 was immobilized to NHS-activated Sepharose 4 FF for subsequent chromatography and batch adsorption experiments. Coupling was achieved through the formation of an amide bond between the amino group of the aminopropyl linker on the ligand and the NHS-activated carboxylic group of the pre-activation resin. The table below shows the results for the coupling reaction of the ligands of Example 2. On average, a fixed ligand density of 15.5 μmol per ml of resin was obtained. The reaction yield averaged 74.5%.

Example 4: Chromatographic Evaluation of Ligands (Example 2)

Experiment

The resins were evaluated in packed mode by microtiter plate chromatography. For each column, about 30 μL of resin was transferred to a 384-well filter plate and sealed with an appropriate top frit. Columns with rProtein A Sepharose FF and Mabsorbent A2P HF were included as controls.

The columns were equilibrated with 6.7 column volumes (cv) of phosphate buffered saline (0.15 M NaCl, 20 mM sodium phosphate, pH 7.3; PBS). Whole IgGs dissolved in 3.3 cv of 0.75 mg ml ⁻¹ , Fab or Fc fragments (all three of which are in PBS) or host cell protein (HCP) dissolved in 0.25 mg ml ⁻¹ Injected. The antibody fragments of Bevacizumab (Fab and Fc) were prepared by digesting whole IgG with papain protease (3) according to the manufacturer's protocol. Unbound protein was washed from the column with 5 cv PBS and then eluted with 5 cv glycine buffer at pH 2.5. The transferred volume was rotated through the column at 50 g for 1-2 minutes. During the injection of the sample, the acceleration was reduced to 10 g and the centrifugation time was increased to 5-10 minutes. Eluate fractions were collected in 384-well plates and protein concentrations were analyzed by a Bradford assay. Protein mass m _i , m _ft and m _e (see below) were calculated as the product of fraction volume and measured protein concentration.

Summary and Results

Binding and elution of several antibodies has been demonstrated. Among them, there were human poly-IgG (h-poly-IgG) mixtures isolated from human serum with three humanized therapeutic antibodies bevacizumab, tocilizumab and palivizumab. In some cases, a mixture of purified Bevacizumab Fab and Fc fragments or rabbit IgG (h-poly-IgG, isolated in serum) was used as additional feed. The selectivity of the ligand for the antibody was determined by examining the binding behavior of the host cell protein. Results for commercially available resins rProtein A Sepharose FF (rProtein A), Mabsorbent A2P (A2P) and MEP Hypercel (Hyper) were included for comparison.

Fraction of the protein, binding (bound) "was calculated by dividing the total weight of the injection the difference between the mass m _ft of the protein detected by the total mass m _i and the flow through (flowthrough) of the injected protein protein.

The 'yield' of the protein was calculated by dividing the mass m _e of the eluted protein by the mass m _i of the injected protein.

"Selectivity (selectivity), of the resin was calculated by dividing the fraction of _HCP B bound fraction (fraction) B _Ab of bound antibodies host cell proteins.

Fractions of 'binding' protein and 'yield' of protein were calculated for each resin based on data for all IgG-antibodies. For bevacizumab fragments and host cell proteins, only a fraction of the 'binding' protein is given. The selectivity of the resin was calculated from data for bevacizumab ex IgG and host cell protein. Because of limited experimental accuracy, selectivity was truncated beyond the value of 10.

Chromatographic results for the resins of Example 3 and the reference resin are given in the table below. Values for L53 are estimates predicted based on the close analogs L49 and L50.

Controls bound all antibodies by nearly 100%. Whereas protein A did not bind any host cell protein (HCP), A2P and Hypercel showed high binding properties to it. The derived selectivity index was 10.0 for Protein A, 1.0 for A2P and 1.3 for Hypercel.

Of the ligands tested in Example 2, L1, L27, L31 and L37 showed the best results with regard to antibody binding and selectivity. All of them bound at least 90% of all human pre IgG antibodies tested, thus having the binding characteristics of a generic Fc-binder. In line with this, L1 and L27 bound the Fc-fragment of Bevacizumab, but not the Fab-fragment. In addition, effective binding of rabbit poly-IgGs to L1 has been demonstrated. The yields of bevacizumab, tocilizumab and palivizumab after chromatography were 87% to 100%. The yield of poly-IgG was slightly lower, 70% to 90%. The selectivity index was 3.3 for L1, 2.9 for L27, 4.4 for L31 and 3.4 for L37.

The other ligands of Example 2 showed a decrease in binding of one or more of the antibodies tested or binding of HCP by at least 40% (derived selectivity index was 2.5 or less).

Example 5: Time-Scale of Isotherms and Couplings

Experiment

Experiments were performed with bevacizumab as an antibody. Parameters of Langmuir isotherm for purified antibodies were determined by batch adsorption experiments in 96-well microtiter plates. In each well, 10 μL of adsorbent slurry (50% v / v) was mixed with 100 μL of protein solution. Initial concentrations were 0.05-5 mg ml ⁻¹ in phosphate buffered saline (0.15 M NaCl, 20 mM phosphate buffer, pH 7.3; PBS). The reaction was stirred vigorously at 25 ° C. for at least 3 hours. The antibody concentration in the supernatants was then measured by the Bradford assay.

Uptake kinetics were similarly investigated by batch adsorption in 96-well filter plates. Again, 10 μL of adsorbent slurry (50% v / v) was mixed with 100 μL of protein solution. However, a fixed initial concentration of 0.75 mg ml ⁻¹ bevacizumab in PBS was used. The reaction was stirred vigorously at 25 ° C. for up to 80 minutes. The supernatants were separated quickly by spinning through the filter after 2.5, 5, 10, 20, 40 and 80 minutes and sampled for analysis. The concentration of the antibody was measured by Bradford assay.

Summary and Results

The subset of the immobilized ligands of Example 3 was characterized in terms of their affinity and maximum capacity for bevacizumab. Moreover, for L1, the timescale required for binding was determined. Commercially available resins rProtein A Sepharose FF (rProtein A) and Mabsorbent A2P (A2P) were included for comparison.

The parameters of the Langmuir isotherm, namely the dissociation constant K _d and the maximum dose q _m , were determined from the concentrations measured in the supernatant. The parameters were estimated by numerical adjustment of the model equation derived from Chase (4).

Absorption kinetics were characterized by the time scale t _0.8 of binding, which was defined as the time after 80% of the equilibrium saturation of the resin by the antibody occurred. To determine t _0.8 , the measured concentrations in the supernatant were interpolated by the adjustment of double-exponentials 5 and the values of t _0.8 were read from the graph. Parameters of Langmuir isotherms and timescales of binding for the reference resin as well as the in-house resin are provided in the table below.

The highest affinity was observed for rProtein A with a K _d of 4.7 μg ml ⁻¹ . The dissociation constants of A2P (49 μg ml ⁻¹ ), Resin L1 (61 μg ml ⁻¹ ) and L27 (92 μg ml ⁻¹ ) were lower by one digit. The dissociation constants of the residual resin were two orders of magnitude lower and ranged from 103 μg ml ⁻¹ (L23) to 252 μg ml ⁻¹ (L17). Regarding the maximum dose, the highest dose was measured at A2P (69 mg per ml of resin). rProtein A had a maximum dose of 41 mg per ml of resin. Maximum doses of the in-house resins were 26 to 37 mg per ml of resin. Absorption of antibodies was fastest in rProtein A (8.9 minutes), followed by A2P (9.3 minutes) and L1 (9.9 minutes).

Example 6 Dynamic Binding Capacity

Experiment

Dynamic binding capacities were determined by purified Bevacizumabro column chromatography. The resins were packed into an analytical column 25 mm long and 3 mm inner diameter. The column was fed with 1 mg ml ^-1 antibody in phosphate buffered saline (150 mM NaCl, 20 mM phosphate buffer, pH 7.3) at a constant flow rate, and the concentration of the antibody in the eluate was monitored online via absorbance 280 nm. The antibody was loaded until the eluent concentration reached a feed concentration of at least 10%. Dynamic binding capacity was determined at a flow rate of 50 cm h ⁻¹ corresponding to 3 minutes of empty column residence time. Equilibrium dose was also determined at a flow rate of 50 cm h ⁻¹ by loading up to complete breakthrough of the antibody. The bound antibody was removed from the column by washing with sodium citrate at pH 3.0 followed by glycine hydrochloride at pH 2.5.

Summary and Results

Dynamic (binding) capacity for bevacizumab as a function of flow rate was determined for L1 (Example 3) and rProtein A Sepharose 4 FF (rProtein A).

'Dynamic Capacity' was defined as the amount of antibody that could be injected onto a column at a constant flow rate until the eluate concentration reached 10% of the feed concentration. This was calculated by multiplying the time t _0.1 after the 10% breakthrough occurred by the flow rate F and the feed concentration c _f .

Equilibrium Capacity is defined as the maximum amount of antibody that binds to a column for a given feed stream concentration. This was calculated by obtaining the following integrals, including feed concentration c _f , eluent concentration c ( t ) and flow rate F over time.

Integral was found numerically. The integration method was limited to the time after complete breakthrough occurred. The calculations of the dynamic and equilibrium capacities were both corrected for the hold-up of the column and the chromatography system. The capacity was normalized by the volume of resin in the column.

The dynamic binding capacity for L1 and rProtein A Sepharose 4 FF as a function of flow rate and the equilibrium binding capacity for the feed concentration of 1 mg ml ⁻¹ bevacizumab are given in the table below.

For rProtein A, a dynamic binding dose of 28.4 mg per ml of resin was determined. The dynamic dose of L1 was 20.2 mg per ml of resin. The calculated equilibrium dose was 40 mg per ml of resin for rProtein A and 25.7 mg per ml of resin for L1.

Example 7: Alkaline Stability

Ligand L1 of Example 2 was tested for its alkaline stability over a period of 8 days. It was treated with 0.1 M or 0.5 M sodium hydroxide at 25 ° C. Hydrolysis was monitored by LC-MS analysis.

At 0.5 M NaOH, the half life of L1 was 288 hours. At 0.1 M NaOH, no degradation of the ligand was detected.

Example 8: Purification of Antibodies from Cell Culture Supernatants

Experiment

The suitability of the resin for purifying the antibody from the cell culture supernatants was evaluated in a mode of filling by microtiter plate chromatography similar to Example 3, or by regular column chromatography. In two cases, bevacizumab added to host cell protein at a concentration of 0.05 mg ml ⁻¹ was used as feed. In addition, chromatographic runs (microtiter plate and column chromatography) of host cell proteins and chromatographic runs of pure antibody alone (only microtiter plate chromatography) were performed. For microtiter plate chromatography, 25 column volumes were injected per run. For column chromatography, 100 column volumes were injected. Columns were equilibrated and washed with PBS before and after injection. The bound protein was eluted with sodium citrate at pH 3.0. Protein concentration in the feed and column eluate was analyzed by Bradford assay or by UV-absorbance at 280 nm.

Summary and Results

Antibodies were purified from cell culture supernatants by chromatography on Resin L1 of Example 3. Chromatography for commercially available resins rProtein A Sepharose FF (rProtein A) and Mabsorbent A2P (A2P) were included for comparison. For microtiter plate chromatography, three chromatography runs under the same conditions were performed for each resin injected with antibodies, host cell proteins or mixtures thereof. For column chromatography, only two runs were performed with the antibody contained in the host cell protein or with the host cell protein alone.

Operation 'yield' after chromatography of the mixture refers to the mass m _{mix, e} of total protein recovered following the elution following injection of the mixture _, the mass m _{HCP, e} recovered after injection of the host cell protein alone and the injected antibody. The mass m of was calculated from _{Ab, i} .

The 'purity' of the antibody after chromatography of the mixture was calculated from the mass m _{mix, e} of total protein recovered following elution following injection of the mixture and the mass m _{HCP, e} recovered after injection of host cell proteins.

Purity and yield obtained after chromatography in Resin L1 and Reference Resin of Example 3 are given in the table below.

Within the experimental accuracy, the yield after microtiter plate chromatography was 100% for rProtein A, 85% for L1 and 12% for A2P. Recovery (12%) from A2P was low due to moderate acidic pH 3.0 at the time of elution. Purity was highest after chromatography on rProtein A (100%), followed by chromatography on Resin L1 (74%). Purity was the lowest after chromatography in A2P, only 32%. The results after column chromatography were in a similar range. Here, rProtein A showed a yield of 97% and a purity of 100%. L1 showed 90% yield and 83% purity.

Documents cited in the Examples:

1. T. Neumann, HD Junker, K. Schmidt, R. Sekul, SPR Based Fragment Screening: Advantages and Applications, Current Topics in Medicinal Chemistry 2007 ; 7: 1630-1642

2. Roth M. Fluorescence reaction for amino acids. Anal Chem 197 ; 43 (7): 880-2.

Rousseaux J, Rousseaux-Prevost R, Bazin H. Optimal conditions for the preparation of Fab and F (ab ') 2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Methods 1983 ; 64 (1-2): 141-146.

4. Chase HA. Prediction of the performance of preparative affinity chromatography. J Chromatogr 1984 ; 297: 179-202.

5. Coffman JL, Kramarczyk JF, Kelley BD. High-throughput screening of chromatographic separations: I. Method development and column modeling. Biotechnology and Bioengineering 2008 ; 100 (4): 605-618.

Claims (15)

For use in affinity purification of an antibody or antibody fragment of a ligand-substituted matrix comprising a support material and one or more ligands covalently attached to the support material,
Use of said ligand is represented by the following formula (I):
Formula (I)

Wherein,
L is the point of attachment of the support material to which the ligand is attached;
Sp is a spacer group;
v is 0 or 1;
Am is an amide group -NR ¹ -C (O)-, wherein NR ¹ is attached to Ar ¹ and -C (O)-is attached to Ar ² , or -C (O)-is attached to Ar ¹ NR ¹ is attached to Ar ² ;
R ¹ is hydrogen or C ₁ to C ₄ alkyl, preferably hydrogen or methyl, more preferably hydrogen;
Ar ¹ is a divalent 5- or 6-membered substituted or unsubstituted aromatic ring;
Ar ² is (a) a 5- or 6-membered heterocyclic aromatic ring attached to a further 5- or 6-membered aromatic ring via a single bond; Or (b) a 5- or 6-membered heterocyclic aromatic ring fused to an additional 5- or 6-membered aromatic ring as part of a polycyclic ring system; Or (c) C ₁ to C ₄ alkyl, C ₂ to C ₄ alkenyl, C ₂ to C ₄ alkynyl, halogen, C ₁ To C ₄ haloalkyl, hydroxyl-substituted C ₁ to C ₄ alkyl, C ₁ to C ₄ alkoxy, hydroxyl-substituted C ₁ to C ₄ alkoxy, C ₁ to C ₄ alkylamino, C ₁ to C ₄ 5- or 6-membered heterocyclic aromatic ring attached to at least one substituent selected from alkylthio, and combinations thereof. The method of claim 1,
Ar ¹ is phenylene, preferably methoxy-substituted phenylene. The method according to claim 1 or 2,
And wherein the C═O and NH groups are bonded to Ar ¹ in a meta position relative to each other. 4. The method according to any one of claims 1 to 3,
5- or 6-membered heterocyclic aromatic ring of Ar ² is characterized in that it is attached to the C═O group via a carbon ring atom adjacent to a ring heteroatom, preferably a nitrogen or oxygen atom. 5. The method according to any one of claims 1 to 4,
5- or 6-membered heterocyclic aromatic ring of Ar ² characterized by containing at least two nitrogen atoms or at least one nitrogen atom and oxygen atom. The method of claim 5,
5- or 6-membered heterocyclic aromatic ring of Ar ² is N-methyl-substituted pyrazole, pyridine, isoxazole or oxadiazole. 8. The method according to any one of claims 1 to 7,
The support material may be carbohydrate or crosslinked carbohydrates, preferably agarose, cellulose, dextran, starch, alginate and carrageenan, sepharose, cefadex; Synthetic polymers, preferably polystyrene, styrene-divinylbenzene copolymers, polyacrylates, PEG-polyacrylate copolymer polymethacrylates, polyvinyl alcohols, polyamides and perfluorocarbons; Inorganics, preferably glass, silica and metal oxides; And a material selected from the composite. 8. The method according to any one of claims 1 to 7,
Said protein is an antibody, preferably an IgG type antibody, or an Fc fusion protein. 9. The method of claim 8,
Said purification is accomplished by binding a ligand of a ligand-substituted matrix to an Fc fragment or domain of an antibody or fusion protein. 10. The method according to claim 8 or 9,
Said Fc fragment or domain or antibody belongs to the group of IgG antibodies, more preferably human IgG or polyclonal or monoclonal IgG of human origin, in particular IgG ₁ , IgG ₂ and IgG ₄ . The ligand-substituted matrix as defined in any one of claims 1 to 7. A method for preparing a ligand-substituted matrix according to claim 11,
A process according to any one of claims 1 to 7, wherein the ligand of formula (I) is attached to the support material. Method for affinity purification of proteins, preferably affinity chromatography,
8. The method of claim 1, wherein the protein to be purified is contacted with a ligand-substituted matrix as defined in any of claims 1-7. The method of claim 13,
Wherein said protein is an antibody or Fc fusion protein, and said ligand binds to the Fc region of the antibody or Fc fusion protein. Ligands represented by the following formula (II):
[Formula (III)]

Wherein,
Sp, Ar ¹ , Ar ² and Am have the meanings as defined in any one of claims 1 to 7.